Microrna compounds and methods for modulating mir-122

ABSTRACT

Described herein are compositions and methods for the inhibition of miR-122 activity. The compositions have certain nucleoside modifications that yield potent inhibitors of miR-122 activity. The compounds may comprise conjugates to facilitate delivery to the liver. The compositions may be administered to subjects infected with hepatitis C virus, as a treatment for hepatitis C virus and related conditions.

This application claims the benefit of U.S. Provisional Application Nos.61/818,432, filed May 1, 2013; 61/822,112, filed May 10, 2013;61/839,550, filed Jun. 26, 2013; 61/895,784, filed Oct. 25, 2013;61/898,704, filed Nov. 1, 2013; and 61/927,897, filed Jan. 15, 2014;each of which is incorporated by reference herein in its entirety forany purpose.

FIELD OF INVENTION

Provided herein are compounds and methods for use in modulating theactivity of miR-122. Such methods comprise treatment of diseases relatedto miR-122 activity, such HCV infection.

DESCRIPTION OF RELATED ART

MicroRNAs (microRNAs), also known as “mature microRNA” are small(approximately 18-24 nucleotides in length), non-coding RNA moleculesencoded in the genomes of plants and animals. In certain instances,highly conserved, endogenously expressed microRNAs regulate theexpression of genes by binding to the 3′-untranslated regions (3′-UTR)of specific mRNAs. More than 1000 different microRNAs have beenidentified in plants and animals. Certain mature microRNAs appear tooriginate from long endogenous primary microRNA transcripts (also knownas pri-microRNAs, pri-mirs, pri-miRs or pri-pre-microRNAs) that areoften hundreds of nucleotides in length (Lee, et al., EMBO J., 2002,21(17), 4663-4670).

miR-122, a microRNA abundantly and specifically expressed in the liver,is a critical host factor for hepatitis C virus accumulation (Jopling etal., Science. 2005, 309(5740), 1577-81). miR-122 interacts with HCV bybinding to two closely spaced seed sequence sites in the 5′ non-codingregion of the HCV genome, resulting in stabilization of the HCV genome,supporting replication and translation (Jangra et al., J Virol., 2010,84: 6615-6625; Machlin, et al., 2011). Importantly, the miR-122 bindingsites are completely conserved in the HCV genome across all genotypesand subtypes (Wilson et al., J. Virol., 2011, 85: 2342-2350) Inhibitionof miR-122 with anti-miR results in reduced total circulatingcholesterol levels in mice and cynomolgus monkey, as well as changes inthe expression of genes involved in cholesterol homeostasis, fatty acid,and lipid metabolism (Esau et al., 2006, Cell Metabolism, 3: 87-98). Inchronically HCV-infected chimpanzees, weekly intravenous administrationof anti-miR to long-lasting and reversible suppression of HCV RNA levelsand reduced total serum cholesterol (Lanford et al., 2010, Science,327:198-201). In chronic treatment naïve HCV infected patients,anti-miR-122 treatment led to a reduction in serum HCV RNA, thusdemonstrating clinical proof-of-concept.

Hepatitis C (HCV) is a hepatotropic RNA virus in the Flaviviridae familyand, addition to causing HCV infection, is a major cause of chronicliver disease and hepatocellular carcinoma. The current standard-of-caretreatment, pegylated interferon in combination with ribavirin, is poorlytolerated by many patients and can have a response rate as low as 50% insome patients. Several direct acting anti-viral NS3 protease inhibitorsare currently approved for use in HCV-infected patients, however theemergence of resistance mutations in HCV requires treatment withadditional agents. Developing therapies include NS3/4A proteaseinhibitors, NS5A protein inhibitors, nucleoside/tide NS5B polymeraseinhibitors and non-nucleoside NS5B inhibitors. However, there remains aneed for additional therapies to treat infected individuals who do notrespond to current treatments, who relapse following successfultreatment, or who have a low tolerability for one or more currently useddrugs. Resistance to antiviral therapy is a major problem associatedwith a high mutation rate of HCV and is seen even with combinations ofdrugs working through multiple mechanisms. Accordingly, therapeuticsthat target conserved, mutation-resistant viral host factors, such asmiR-122, represent an opportunity to effect higher and more durable curerates.

SUMMARY OF INVENTION

Provided herein are compounds comprising a modified oligonucleotideconsisting of 16 to 22 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-122(SEQ ID NO: 1) and wherein the modified oligonucleotide comprises atleast 16 contiguous nucleosides of the following nucleoside pattern I inthe 5′ to 3′ orientation:

(R)_(X)-N^(Q)-N^(Q)-N^(B)-N^(B)-N^(Q)-N^(B)-N^(Q)-N^(B)-N^(Q)-N^(B)-N^(B)-(N^(Z))_(Y)

-   -   wherein each R is, independently, a non-bicyclic nucleoside or a        bicyclic nucleoside;    -   X is from 4 to 10;    -   each N^(B) is, independently, a bicyclic nucleoside;    -   each N^(Q) is, independently, a non-bicyclic nucleoside;    -   Y is 0 or 1; and    -   N^(Z) is a modified nucleoside or an unmodified nucleoside.

In certain embodiments, a compound provided herein comprises a modifiedoligonucleotide comprising at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, or 22 contiguous nucleosides ofnucleoside pattern I.

In certain embodiments, each bicyclic nucleoside is independentlyselected from an LNA nucleoside, a cEt nucleoside, and an ENAnucleoside. In certain embodiments, at least two bicyclic nucleosidesare different from one another. In certain embodiments, all bicyclicnucleosides have the same sugar moiety as one another. In certainembodiments, each bicyclic nucleoside is a cEt nucleoside. In certainembodiments, a cEt nucleoside is an S-cEt nucleoside. In certainembodiments, a cEt nucleoside is an R-cEt nucleoside. In certainembodiments, each bicyclic nucleoside is an LNA nucleoside.

In certain embodiments, at least two non-bicyclic nucleosides comprisesugar moieties that are different from one another. In certainembodiments, each non-bicyclic nucleoside has the same type of sugarmoiety. In certain embodiments, each non-bicyclic nucleoside isindependently selected from a β-D-deoxyribonucleoside, aβ-D-ribonucleoside, 2′-O-methyl nucleoside, a 2′-O-methoxyethylnucleoside, and a 2′-fluoronucleoside. In certain embodiments, eachnon-bicyclic nucleoside is independently selected from aβ-D-deoxyribonucleoside, and a 2′-O-methoxyethyl nucleoside. In certainembodiments, each non-bicyclic nucleoside is a β-D-deoxyribonucleoside.In certain embodiments, each non-bicyclic nucleoside is a 2′-MOEnucleoside. In certain embodiments, no more than two non-bicyclicnucleosides are 2′-MOE nucleosides, wherein each other non-bicyclicnucleoside is a β-D-deoxyribonucleoside. In certain embodiments, the5′-most and the 3′-most non-bicyclic nucleosides are 2′-MOE nucleosidesand each other non-bicyclic nucleoside is a β-D-deoxyribonucleoside. Incertain embodiments, two non-bicyclic nucleosides are 2′-MOE nucleosidesand each other non-bicyclic nucleoside is a β-D-deoxyribonucleoside.

In certain embodiments, each R is a 2′-MOE nucleoside. In certainembodiments, X is 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, Y is0. In certain embodiments, Y is 1.

In certain embodiments, X is 7, each R is a 2′-O-methoxyethylnucleoside, each N^(B) is an S-cEt nucleoside, each N^(Q) is aβ-D-deoxyribonucleoside, and Y is 0.

In certain embodiments, X is 4; (R)_(X) is N^(R1)-N^(R2)-N^(R3)-N^(R4),wherein each of N^(R1) and N^(R3) is a S-cEt nucleoside and each ofN^(R2) and N^(R4) is a β-D-deoxyribonucleoside; each N^(B) is an S-cEtnucleoside; each N^(Q) is β-D-deoxyribonucleoside; Y is 1; and N^(Z) isa β-D-deoxyribonucleoside.

In certain embodiments, X is 4; (R)_(X) is N^(R1)-N^(R2)-N^(R3)-N^(R4),wherein each of N^(R1) and N^(R4) is a S-cEt nucleoside and each ofN^(R2) and N^(R3) is a β-D-deoxyribonucleoside; each N^(B) is an S-cEtnucleoside; each N^(Q) is β-D-deoxyribonucleoside; Y is 1; and N^(Z) isa 2′-O-methoxyethyl nucleoside.

In certain embodiments, X is 7; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), and N^(R4) and is a 2′-O-methoxyethylnucleoside, each of N^(R5) and N^(R7) is a β-D-deoxyribonucleoside, andN^(R6) is S-cEt nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, X is 7; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), N^(R4), and N^(R5) is a 2′-O-methoxyethylnucleoside, N^(R6) is S-cEt nucleoside, and N^(R7) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each NQ is aβ-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, X is 7; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is 2′-O-methoxyethylnucleoside, and N^(R7) is a β-D-deoxyribonucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, X is 10; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7)-N^(R8)-N^(R9)-N^(R10),wherein each of N^(R1),N^(R2),N^(R3), N^(R4),N^(R5), and N^(R6) is2′-O-methoxyethyl nucleoside, each of N^(R7) and N^(R9) is a an S-cEtnucleoside; each of N^(R8) and NR¹⁰ is a β-D-deoxyribonucleoside; eachN^(B) is an S-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside;and Y is 0.

In certain embodiments, X is 10; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7)-N^(R8)-N^(R9)-N^(R10),wherein each of N^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is2′-O-methoxyethyl nucleoside, each of N^(R7) and N^(R9) is a an S-cEtnucleoside; and each of N^(R8) and N^(R10) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; Y is 1 and N_(Z) is a 2′-O-methoxyethylnucleoside.

In certain embodiments, X is 4; (R)_(X) is N^(R1)-N^(R2)-N^(R3)-N^(R4),wherein each of N^(R1) and N^(R4) is an S-cEt nucleoside, and each ofN^(R1) and N^(R3) is a β-D-deoxyribonucleoside; each N^(B) is an S-cEtnucleoside; each N^(Q) is a β-D-deoxyribonucleoside; Y is 1 and N^(Z) isa β-D-deoxyribonucleoside.

In certain embodiments, X is 4; (R)_(X) is N^(R1)-N^(R2)-N^(R3)-N^(R4),wherein N^(R1) is a 2′-O-methoxyethyl nucleoside, each of N^(R2) andN^(R4) is an S-cEt nucleoside, and N^(R3) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each NQ is a β-D-deoxyribonucleoside;Y is 1 and N^(Z) is a 2′-O-methoxyethyl nucleoside.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 90%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% complementary to the nucleobase sequence of miR-122 (SEQ IDNO: 1).

In certain embodiments, wherein at least one internucleoside linkage isa modified internucleoside linkage, or wherein each internucleosidelinkage is a modified internucleoside linkage, and, optionally, whereinthe modified internucleoside linkage is a phosphorothioateinternucleoside linkage.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is selected from SEQ ID NOs: 3 to 6, wherein each T isindependently selected from T and U.

In certain embodiments, the modified oligonucleotide has 0, 1, 2, or 3mismatches with respect to the nucleobase sequence of miR-122.

In certain embodiments a compound has the structure:

-   -   A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)        ^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) (SEQ ID        NO: 4);    -   C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A (SEQ ID NO: 3);    -   ^(Me)C_(S)CAT_(S)TGT_(S)        ^(Me)C_(S)A^(Me)C_(S)A^(Me)C_(S)T^(Me)C_(S) ^(Me)C_(S)A_(E) (SEQ        ID NO: 3);    -   A_(E) ^(Me)C_(E)A_(E)        ^(Me)C_(E)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) (SEQ ID NO:        4);    -   A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)        ^(Me)C_(E)A_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) (SEQ ID NO:        4);    -   A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)        ^(Me)C_(E)A_(E)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) (SEQ ID NO:        4);    -   ^(Me)C_(E)A_(E)A_(E)A_(E)        ^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)        (SEQ ID NO: 5);    -   ^(Me)C_(E)A_(E)A_(E)A_(E)        ^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E)        (SEQ ID NO: 6);    -   C_(S)CAU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A (SEQ ID NO: 3);        or    -   ^(Me)C_(E)C_(S)AU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A_(E)        (SEQ ID NO: 3);        wherein the superscript “Me” indicates 5-methylcytosine;        nucleosides not followed by a subscript are        (3-D-deoxyribonucleosides; nucleosides followed by a subscript        “E” are 2′-MOE nucleosides; nucleosides followed by a subscript        “S” are S-cEt nucleosides; and each internucleoside linkage is a        phosphorothioate internucleoside linkage.

In some embodiments, a compound has the structure:

-   -   U_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A_(S); or    -   C_(S)A_(S)C_(S)A_(S)C_(S)U_(S)C_(S)C_(S)        wherein nucleosides not followed by a subscript are        β-D-deoxyribonucleosides; nucleosides followed by a subscript        “S” are S-cEt nucleosides; and each internucleoside linkage is a        phosphorothioate internucleoside linkage. In some such        embodiments, the compound is compound 38591, 38633, 38998, or        38634.

Any of the compounds provided herein may comprise a conjugate moietylinked to the 5′ terminus or the 3′ terminus of the modifiedoligonucleotide. In certain embodiments, the compound comprises aconjugate moiety linked to the 3′ terminus of the modifiedoligonucleotide. In certain embodiments, the compound comprises aconjugate moiety linked to the 5′ terminus of the modifiedoligonucleotide. In certain embodiments, the compound comprises a firstconjugate moiety linked to the 3′ terminus of the modifiedoligonucleotide and a second conjugate moiety linked to the 5′ terminusof the modified oligonucleotide. In certain embodiments, the conjugatemoiety comprises at least one ligand selected from a carbohydrate,cholesterol, a lipid, a phospholipid, an antibody, a lipoprotein, ahormone, a peptide, a vitamin, a steroid, and a cationic lipid.

In certain embodiments, a compound has the structure L_(n)-linker-MO,wherein each L is, independently, a ligand and n is from 1 to 10; and MOis a modified oligonucleotide.

In certain embodiments, a compound has the structure L_(n)-linker-X-MO,wherein each L is, independently, a ligand and n is from 1 to 10; X is aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide.

In certain embodiments, a compound has the structureL_(n)-linker-X₁-N_(m)-X₂-MO, wherein each L is, independently, a ligandand n is from 1 to 10; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; X₁ and X₂ are each,independently, a phosphodiester linkage or a phosphorothioate linkage;and MO is a modified oligonucleotide.

In certain embodiments, a compound has the structureL_(n)-linker-X-N_(m)-Y-MO, wherein each L is, independently, a ligandand n is from 1 to 10; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; X is a phosphodiesterlinkage or a phosphorothioate linkage; Y is a phosphodiester linkage;and MO is a modified oligonucleotide.

In certain embodiments, a compound has the structureL_(n)-linker-Y-N_(m)-Y-MO, wherein each L is, independently, a ligandand n is from 1 to 10; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; each Y is a phosphodiesterlinkage; and MO is a modified oligonucleotide.

In certain embodiments, if n is greater than 1, L_(n)-linker has thestructure:

wherein each L is, independently, a ligand; n is from 1 to 10; S is ascaffold; and Q′ and Q″ are, independently, linking groups.

In certain embodiments, Q′ and Q″ are each independently selected from apeptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl, asubstituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments, a scaffold links 2, 3, 4, or 5 ligands to amodified oligonucleotide. In certain embodiments, a scaffold links 3ligands to a modified oligonucleotide.

A nonlimiting exemplary Structure E is Structure E(i):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, and R₄are each, independently, selected from H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, and R₄ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments, R₁, R₂, R₃, and R₄ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(ii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁ is selected fromH, C₁-C₆ alkyl, and substituted C₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁ is selected from H, methyl, ethyl, propyl,isopropyl, and butyl. In some embodiments, R₁ is H or methyl.

A further nonlimiting exemplary Structure E is Structure E(iii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, R₄, andR₅ are each, independently, selected from H, C₁-C₆ alkyl, andsubstituted C₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, R₄, and R₅ are each,independently, selected from H, methyl, ethyl, propyl, isopropyl, andbutyl. In some embodiments R₁, R₂, R₃, R₄, and R₅ are each selected fromH and methyl.

A further nonlimiting exemplary Structure E is Structure E(iv):

wherein L₁ and L₂ are each, independently, a ligand; Q′₁, Q′₂, and Q″are each, independently, a linking group; and R₁, R₂, and R₃ are each,independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, and Q″ are each, independently, selectedfrom a peptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl,a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid. In someembodiments, R₁, R₂, and R₃ are each, independently, selected from H,methyl, ethyl, propyl, isopropyl, and butyl. In some embodiments R₁, R₂,and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(v):

wherein L₁ and L₂ are each, independently, a ligand; Q′₁, Q′₂, and Q″are each, independently, a linking group; and R₁, R₂, and R₃ are each,independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, and Q″ are each, independently, selectedfrom a peptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl,a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid. In someembodiments, R₁, R₂, and R₃ are each, independently, selected from H,methyl, ethyl, propyl, isopropyl, and butyl. In some embodiments R₁, R₂,and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(vi):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, and R₃ areeach, independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, and R₃ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(vii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; R₁, R₂, and R₃ areeach, independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl; and Z and Z′ are each independently selected from O and S.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, and R₃ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, and R₃ are each selected from H and methyl. In someembodiments, Z or Z′ on at least one P atom is S, and the other Z or Z′is O (i.e., a phosphorothioate linkage). In some embodiments, each—OP(Z)(Z′)O— is a phosphorothioate linkage. In some embodiments, Z andZ′ are both O on at least one P atom (i.e., a phosphodiester linkage).In some embodiments, each —OP(Z)(Z′)O— is a phosphodiester linkage.

A further nonlimiting exemplary Structure E is Structure E(viii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, and R₄are each, independently, selected from H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, and R₄ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, R₃, and R₄ are each selected from H and methyl.

Nonlimiting exemplary scaffolds and/or linkers comprising scaffolds, andsynthesis thereof, are described, e.g., PCT Publication No. WO2013/033230, U.S. Pat. No. 8,106,022B2, U.S. Publication No.2012/0157509 A1; U.S. Pat. No. 5,994,517; U.S. Pat. No. 7,491,805B2;U.S. Pat. No. 8,313,772B2; Manoharan, M., Chapter 16, Antisense DrugTechnology, Crooke, S. T., Marcel Dekker, Inc., 2001, 391-469.

In certain embodiments, a compound has the structure:

wherein:

-   -   B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,        —Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—;    -   MO is a modified oligonucleotide;    -   R^(N) is selected from H, methyl, ethyl, propyl, isopropyl,        butyl, and benzyl;    -   Z, Z′, and Z″ are each independently selected from O and S;    -   each N is, independently, a modified or unmodified nucleoside;    -   m is from 1 to 5;    -   X is selected from a phosphodiester linkage and a        phosphorothioate linkage;    -   Y is a phosphodiester linkage; and    -   the wavy line indicates the connection to the rest of the linker        and ligand(s).

In certain embodiments, X is a phosphodiester linkage.

In certain embodiments, n is from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Incertain embodiments, n is 3.

In certain embodiments, at least one ligand is a carbohydrate.

In certain embodiments, at least one ligand is selected from mannose,glucose, galactose, ribose, arabinose, fructose, fucose, xylose,D-mannose, L-mannose, D-galactose, L-galactose, D-glucose, L-glucose,D-ribose, L-ribose, D-arabinose, L-arabinose, D-fructose, L-fructose,D-fucose, L-fucose, D-xylose, L-xylose, alpha-D-mannofuranose,beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose,alpha-D-glucofuranose, Beta-D-glucofuranose, alpha-D-glucopyranose,beta-D-glucopyranose, alpha-D-galactofuranose, beta-D-galactofuranose,alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-ribofuranose,beta-D-ribofuranose, alpha-D-ribopyranose, beta-D-ribopyranose,alpha-D-fructofuranose, alpha-D-fructopyranose, glucosamine,galactosamine, sialic acid, N-acetylgalactosamine.

In certain embodiments, at least one ligand is selected fromN-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, andN-iso-butanoyl-galactosamine

In certain embodiments, each ligand is N-acetylgalactosamine.

In certain embodiments, a compound has the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide.

In certain embodiments, a compound provided herein comprises a modifiednucleotide and a conjugate moiety, wherein the modified oligonucleotidehas the structure C_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) (SEQID NO: 7), wherein the subscript “L” indicates an LNA and nucleosidesnot followed by a subscript are β-D-deoxyribonucleosides, and eachinternucleoside linkage is a phosphorothioate internucleoside linkage,and wherein the conjugate moiety is linked to the 3′ terminus of themodified oligonucleotide and has the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide.

In certain embodiments, at least one of X₁ and X₂ is a phosphodiesterlinkage. In certain embodiments, each of X₁ and X₂ is a phosphodiesterlinkage. In certain embodiments, m is 1. In certain embodiments, m is 2,3, 4, or 5.

In certain embodiments, N_(m) is N′_(p)N“, wherein each N′ is,independently, a modifid or unmodified nucleoside and p is from 0 to 4;and N” is a nucleoside comprising an unmodified sugar moiety. In certainembodiments, p is 0. In certain embodiments, p is 1, 2, 3, or 4.

In certain embodiments, each N′ comprises an unmodified sugar moiety. Incertain embodiments, each unmodified sugar moiety is, independently, aβ-D-ribose or a β-D-deoxyribose. In certain embodiments, N″ comprises apurine nucleobase. In certain embodiments, N″ comprises a pyrimidinenucleobase. In certain embodiments, at least one N′ comprises a purinenucleobase. In certain embodiments, each purine nucleobase isindependently selected from adenine, guanine, hypoxanthine, xanthine,and 7-methylguanine. In certain embodiments, N″ is aβ-D-deoxyriboadenosine or a β-D-deoxyriboguanosine. In certainembodiments, at least one N′ comprises a pyrimidine nucleobase. Incertain embodiments, each pyrimidine nucleobase is independentlyselected from cytosine, 5-methylcytosine, thymine, uracil, and5,6-dihydrouracil.

In any of the embodiments described herein, the sugar moiety of each Nis independently selected from a β-D-ribose, a β-D-deoxyribose, a2′-O-methoxy sugar, a 2′-O-methyl sugar, a 2′-fluoro sugar, and abicyclic sugar moiety. In certain embodiments, each bicyclic sugarmoiety is independently selected from a cEt sugar moiety, an LNA sugarmoiety, and an ENA sugar moiety. In certain embodiments, a cEt sugarmoiety is an S-cEt sugar moiety. In certain embodiments, a cEt sugarmoiety is an R-cEt sugar moiety. In any embodiments described herein,the sugar moiety of each N may be independently selected fromβ-D-ribose, a β-D-deoxyribose, and a 2′-fluoro sugar.

Provided herein are compounds comprising a modified nucleotide and aconjugate moiety, wherein the modified oligonucleotide has the structureA_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) (SEQ ID NO: 4),wherein nucleosides not followed by a subscript areβ-D-deoxyribonucleosides, nucleosides followed by a subscript “E” are2′-MOE nucleosides, nucleosides followed by a subscript “S” are S-cEtnucleosides, and each internucleoside linkage is a phosphorothioateinternucleoside linkage; and wherein the conjugate moiety is linked tothe 3′ terminus of the modified oligonucleotide and has the structure:

wherein X is a phosphodiester linkage; m is 1; N is aβ-D-deoxyriboadenosine; Y is a phosphodiester linkage; and MO is themodified oligonucleotide.

Provided herein are compounds comprising a modified nucleotide and aconjugate moiety, wherein the modified oligonucleotide has the structureC_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) (SEQ ID NO: 7), whereinthe subscript “L” indicates an LNA and nucleosides not followed by asubscript are β-D-deoxyribonucleosides, and each internucleoside linkageis a phosphorothioate internucleoside linkage, and wherein the conjugatemoiety is linked to the 3′ terminus of the modified oligonucleotide andhas the structure:

wherein X is a phosphodiester linkage; m is 1; N is aβ-D-deoxyriboadenosine; Y is a phosphodiester linkage; and MO is themodified oligonucleotide. In some embodiments, all of the C_(L)nucleosides are ^(Me)C_(L) nucleosides, wherein the superscript “Me”indicates 5-methylcytosine.

Provided herein are methods of inhibiting the activity of miR-122 in acell comprising contacting a cell with any compound provided herein. Incertain embodiments, the cell is cell is in vivo. In certainembodiments, cell is in vitro.

Provided herein are methods of administering to an HCV-infected subjectany of the compounds provided herein. In certain embodiments, theadministering reduces the symptoms of HCV infection. In certainembodiments, the administering prevents a rebound in serum HCV RNA. Incertain embodiments, the administering delays a rebound in serum HCVRNA. In certain embodiments, a subject having HCV infection is selectedfor treatment with a compound provided herein. In certain embodiments,an HCV-infected subject is infected with one or more HCV genotypesselected from genotype 1, genotype 2, genotype 3, genotype 4, genotype5, and genotype 6. In certain embodiments, prior to administration of acompound provided herein, the subject was determined to be infected withone or more HCV genotypes selected from genotype 1, genotype 2, genotype3, genotype 4, genotype 5, and genotype 6. In certain embodiments, theHCV genotype is selected from genotype 1a, genotype 1b, genotype 2a,genotype 2b, genotype 2c, genotype 2d, genotype 3a, genotype 3b,genotype 3c, genotype 3d, genotype 3e, genotype 3f, genotype 4a,genotype 4b, genotype 4c, genotype 4d, genotype 4e, genotype 4f,genotype 4g, genotype 4h, genotype 4i, genotype 4j, genotype 5a, andgenotype 6a. In certain embodiments, the HCV genotype is selected fromgenotype 1a, 1b, and 2.

Any of the methods provided here may comprise administering at least oneadditional therapeutic agent. In certain embodiments, the at least onetherapeutic agent is selected from a protease inhibitor, a polymeraseinhibitor, a cofactor inhibitor, an RNA polymerase inhibitor, astructural protein inhibitor, a non-structural protein inhibitor, acyclophilin inhibitor, an entry inhibitor, a TLR7 agonist, and aninterferon. In certain embodiments, the at least one therapeutic agentis selected from a protease inhibitor, an NS5A inhibitor, an NS3/4Ainhibitor, a nucleoside NS5B inhibitor, a nucleotide NS5B inhibitor, anon-nucleoside NS5B inhibitor, a cyclophilin inhibitor and aninterferon. In certain embodiments, the at least one therapeutic agentis selected from interferon alfa-2a, interferon alpha-2b, interferonalfacon-1, peginterferon alpha-2b, peginterferon alpha-2a,interferon-alpha-2b extended release, interferon lambda, sofosbuvir,ribavirin, telapravir, boceprevir, vaniprevir, asunaprevir, ritonavir,setrobuvir, daclastavir, simeprevir, alisporivir, mericitabine,tegobuvir, danoprevir, sovaprevir, and neceprevir. In certainembodiments, the at least one therapeutic agent is selected from aninterferon, ribavirin, and telapravir.

In certain embodiments, a subject is infected with an HCV variant thatis resistant to at least one therapeutic agent. In certain embodiments,a subject is infected with an HCV variant that is resistant to adirect-acting anti-viral agent. In certain embodiments, a subject isinfected with an HCV variant that is resistant to at least onetherapeutic agent selected from a protease inhibitor, a polymeraseinhibitor, a cofactor inhibitor, an RNA polymerase inhibitor, astructural protein inhibitor, a non-structural protein inhibitor, and acyclophilin inhibitor. In certain embodiments, a subject is infectedwith an HCV variant that is resistant to at least one therapeutic agentselected from a protease inhibitor, an NS5A inhibitor, an NS3/4Ainhibitor, a nucleoside NS5B inhibitor, a nucleotide NS5B inhibitor, anon-nucleoside NS5B inhibitor, and a cyclophilin inhibitor. In certainembodiments, a subject is infected with an HCV variant that is resistantto at least one therapeutic agent selected from sofosbuvir, ribavirin,telapravir, boceprevir, vaniprevir, asunaprevir, ritonavir, setrobuvir,daclastavir, simeprevir, alisporivir, mericitabine, tegobuvir,danoprevir, sovaprevir, and neceprevir.

In certain embodiments, an HCV-infected subject is a non-responder to atleast one therapeutic agent. In certain embodiments, an HCV-infectedsubject is an interferon non-responder. In certain embodiments, anHCV-infected subject is a direct-acting anti-viral non-responder.

Any of the methods provided herein may comprise selecting a subjecthaving a HCV RNA level greater than 350,000 copies per milliliter ofserum. In certain embodiments, a subject has an HCV RNA level between350,000 and 3,500,000 copies per milliliter of serum. In certainembodiments, a subject has an HCV RNA level greater than 3,500,000copies per milliliter of serum.

In certain embodiments, an HCV-infected subject has an HCV-associateddisease. In certain embodiments, an HCV-associated disease is cirrhosis,liver fibrosis, steatohepatitis, steatosis, or hepatocellular carcinoma.

In certain embodiments, an HCV-infected subject has one or more diseasesthat are not HCV-associated diseases. In certain embodiments, anHCV-infected subject is infected with one or more viruses other thanHCV. In certain embodiments, an HCV-infected subject is infected withhuman immunodeficiency virus (HIV). In certain embodiments, the methodsprovided herein comprise administering an additional therapeutic agentis an anti-viral agent used in the treatment of HIV infection. Incertain embodiments, an additional therapeutic agent is a non-nucleosidereverse transcriptase inhibitors (NNRTIs). In certain embodiments, anadditional therapeutic agent is a nucleoside reverse transcriptaseinhibitors (NRTIs). In certain embodiments, an additional therapeuticagent is a protease inhibitor. In certain embodiments, an additionaltherapeutic agent is an entry inhibitor or fusion inhibitor. In certainembodiments, an additional therapeutic agent is an integrase inhibitor.In certain embodiments, an additional therapeutic agent is selected fromefavirenz, etravirine, nevirapine, abacavir, emtricitabine, tenofovir,lamivudine, zidovudine, atazanavir, darunavir, fosamprenavir, ritonavir,enfuvirtide, maraviroc, and raltegravir.

Any of the methods provided herein may comprise administering a dose ofthe compound sufficient to reduce HCV RNA level. In certain embodiments,the administered dose of the compound reduces HCV RNA level below 40copies per ml of serum. In certain embodiments, the administered dose ofthe compound achieves at least a 2-log reduction in HCV RNA level. Incertain embodiments, administering a compound provided herein achieves asustained virological response. In certain embodiments, the administereddose of the compound is sufficient to achieve an HCV RNA level reductionof at least 0.5 fold, at least 1.0 fold, at least 1.5 fold, at least 2.0fold, or at least 2.5 fold. In certain embodiments, the HCV RNA levelreduction is achieved after two weeks, three weeks, four weeks, fiveweeks, or six weeks of a first administration of the compound. Incertain embodiments, a compound provided herein is administered once perweek, once per two weeks, once per three weeks, once per four weeks, oronce per month. In certain embodiments, a compound provided herein isadministered once per two months or once per three months. In someembodiments, a compound provided herein is administered once per fourweeks.

In certain embodiments, the dose of the compound administered is lessthan or equal to 5 mg/kg per week, less than or equal to 5 mg/kg, lessthan or equal to 4.5 mg/kg, less than or equal to 4.0 mg/kg, less thanor equal to 3.5 mg/kg, less than or equal to 3.0 mg/kg, less than orequal to 2.5 mg/kg, less than or equal to 2.0 mg/kg, less than or equalto 1.5 mg/kg, or less than or equal to 1.0 mg/kg. In certainembodiments, the compound is administered at a dose within a range of 1to 5 mg/kg, or 1 to 4 mg/kg, or 2 to 5 mg/kg, or 2 to 4 mg/kg. Incertain embodiments, the dose of the compound administered is less thanor equal to 10 mg/kg, less than or equal to 7.5 mg/kg, less than orequal to 10 mg/kg per week, or less than or equal to 7.5 mg/kg per week.

In certain embodiments, administration of a compound provided hereinnormalizes liver enzyme levels, wherein the liver enzyme is optionallyalanine aminotransferase.

In any of the embodiments provided herein, the compound is present in apharmaceutical composition.

Provided herein are compounds for use in treating an HCV-infectedsubject.

In certain embodiments, a subject is a human.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B. In vivo potency of anti-miR-122 modifiedoligonucleotides. (A) Onset and duration of action of anti-miR-122,following a single administration of compound at the indicated doses.(B) De-repression of ALDOA seven days after a single dose ofanti-miR-122 compound at the indicated doses.

FIG. 2. Structure of a conjugate moiety comprising three GalNAc ligands.

FIG. 3A, 3B, and 3C. Conjugated modified oligonucleotide structures.

FIG. 4A, 4B, and 4C. In vivo potency of GalNAc-conjugated anti-miR-122modified oligonucleotides.

FIGS. 5A and 5B. Antisense inhibition of miR-122 reduces HCV titer.

FIGS. 6A and 6B. In vivo potency of GalNAc-conjugated anti-miR-122modified oligonucleotides.

FIGS. 7A and 7B. In vivo potency of GalNAc-conjugated anti-miR-122modified oligonucleotides.

FIGS. 8A and 8B. In vivo potency of GalNAc-conjugated anti-miR-122modified oligonucleotides.

FIGS. 9A and 9B. Pharmacokinetics of anti-miR-122 compounds.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in thearts to which the invention belongs. Unless specific definitions areprovided, the nomenclature utilized in connection with, and theprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. In the event thatthere is a plurality of definitions for terms herein, those in thissection prevail. Standard techniques may be used for chemical synthesis,chemical analysis, pharmaceutical preparation, formulation and delivery,and treatment of subjects. Certain such techniques and procedures may befound for example in “Carbohydrate Modifications in Antisense Research”Edited by Sangvi and Cook, American Chemical Society, Washington D.C.,1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., 18th edition, 1990; and which is hereby incorporated byreference for any purpose. Where permitted, all patents, patentapplications, published applications and publications, GENBANKsequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. Where reference is made to a URL orother such identifier or address, it is understood that such identifierscan change and particular information on the internet can change, butequivalent information can be found by searching the internet. Referencethereto evidences the availability and public dissemination of suchinformation.

Before the present compositions and methods are disclosed and described,it is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

DEFINITIONS

“HCV infection” means infection with one or more genotypes of theHepatitis C Virus. “HCV-infected subject” means a subject who has beeninfected with one or more genotypes of the hepatitis C virus. AnHCV-infected subject may or may not exhibit symptoms of HCV infection.HCV-infected subjects include subjects who have been infected with oneor more genotypes of HCV, but HCV RNA in the blood of the subject isbelow detectable levels.

“HCV-associated disease” means a pathological process that is mediatedby HCV infection. HCV-associated diseases include, but are not limitedto, cirrhosis, liver fibrosis, steatoheptatitis, and hepatocellularcarcinoma.

“Blood HCV RNA” means hepatitis C virus RNA present in the blood of anHCV-infected subject. Blood includes whole blood and serum.

“Rebound in serum HCV RNA” means an increase in HCV RNA level followinga previous decrease in HCV RNA level.

“HCV RNA level” means the amount of HCV RNA in a given volume of theblood of a subject. HCV RNA level may be expressed as copies of RNA permilliliter. “HCV RNA level” may also be called “HCV viral load” or “HCVRNA titer.”

“Sustained virological response” means undetectable hepatitis C virusRNA in the blood of the subject at the end of an entire course oftreatment and after a further six months. In certain embodiments, HCVRNA is considered undetectable below 40 copies per milliliter of blood.

“Non-responder” means a subject who has received treatment but is notexperiencing a clinically acceptable improvement in disease markers orsymptoms.

“Interferon non-responder” means an HCV-infected subject who hasreceived treatment with interferon, but is not experiencing a clinicallyacceptable reduction in HCV RNA level.

“Direct-acting anti-viral agent” means a pharmaceutical agent thatinhibits the activity of an HCV enzyme.

“Direct-acting anti-viral non-responder” means an HCV-infected subjectwho has received treatment with a direct-acting anti-viral agent, but isnot experiencing a clinically acceptable reduction in HCV RNA level. Incertain embodiments, the virus has developed resistance to thedirect-acting anti-viral agent.

“miR-122-associated condition” means any disease, disorder or conditionthat can be treated, prevented or ameliorated by modulating miR-122. AmiR-122-associated disease need not be characterized by excess miR-122.miR-122-associated diseases included, without limitation, HCV infection,elevated cholesterol, and iron overload disorders.

“Iron overload disorder” means any disease, disorder or conditioncharacterized by excess iron in the body. “Subject” means a human ornon-human animal selected for treatment or therapy.

“Subject in need thereof′ means a subject that is identified as in needof a therapy or treatment.

“Subject suspected of having” means a subject exhibiting one or moreclinical indicators of a disease.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, andintramuscular administration.

“Subcutaneous administration” means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Administered concomitantly” refers to the co-administration of two ormore agents to a subject in any manner in which the pharmacologicaleffects of each agent are present in a subject. Concomitantadministration does not require that both agents be administered in asingle pharmaceutical composition, in the same dosage form, or by thesame route of administration. The effects of both agents need not bepresent at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Duration” means the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent orpharmaceutical composition are administered.

“Therapy” means a disease treatment method. In certain embodiments,therapy includes, but is not limited to, chemotherapy, radiationtherapy, or administration of a pharmaceutical agent.

“Treatment” means the application of one or more specific proceduresused for the cure or amelioration of a disease. In certain embodiments,the specific procedure is the administration of one or morepharmaceutical agents.

“Amelioration” means a lessening of severity of at least one indicatorof a condition or disease. In certain embodiments, amelioration includesa delay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“At risk for developing” means the state in which a subject ispredisposed to developing a condition or disease. In certainembodiments, a subject at risk for developing a condition or diseaseexhibits one or more symptoms of the condition or disease, but does notexhibit a sufficient number of symptoms to be diagnosed with thecondition or disease. In certain embodiments, a subject at risk fordeveloping a condition or disease exhibits one or more symptoms of thecondition or disease, but to a lesser extent required to be diagnosedwith the condition or disease.

“Prevent the onset of means to prevent the development of a condition ordisease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Delay the onset of means to delay the development of a condition ordisease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Therapeutic agent” means a pharmaceutical agent used for the cure,amelioration or prevention of a disease.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration. In certain embodiments, a dose may beadministered in two or more boluses, tablets, or injections. Forexample, in certain embodiments, where subcutaneous administration isdesired, the desired dose requires a volume not easily accommodated by asingle injection. In such embodiments, two or more injections may beused to achieve the desired dose. In certain embodiments, a dose may beadministered in two or more injections to minimize injection sitereaction in an individual. In certain embodiments, a dose isadministered as a slow infusion.

“Dosage unit” means a form in which a pharmaceutical agent is provided.In certain embodiments, a dosage unit is a vial containing lyophilizedoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted oligonucleotide.

“Therapeutically effective amount” refers to an amount of apharmaceutical agent that provides a therapeutic benefit to an animal.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical composition may comprise a sterile aqueoussolution.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Improved organ function” means a change in organ function toward normallimits. In certain embodiments, organ function is assessed by measuringmolecules found in a subject's blood or urine. For example, in certainembodiments, improved liver function is measured by a reduction in bloodliver transaminase levels. In certain embodiments, improved kidneyfunction is measured by a reduction in blood urea nitrogen, a reductionin proteinuria, a reduction in albuminuria, etc.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits.

“Side effect” means a physiological response attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies. Suchside effects may be detected directly or indirectly. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Injection site reaction” means inflammation or abnormal redness of skinat a site of injection in an individual.

“Subject compliance” means adherence to a recommended or prescribedtherapy by a subject.

“Comply” means the adherence with a recommended therapy by a subject.

“Recommended therapy” means a treatment recommended by a medicalprofessional to treat, ameliorate, delay, or prevent a disease.

“miR-122” means a microRNA having the nucleobase sequenceUGGAGUGUGACAAUGGUGUUUG (SEQ ID NO: 1).

“miR-122 stem-loop” means the microRNA precursor having the nucleobasesequence CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC (SEQ ID NO: 2).

“Anti-miR” means an oligonucleotide having a nucleobase sequencecomplementary to a microRNA. In certain embodiments, an anti-miR is amodified oligonucleotide.

“Anti-miR-122” means an oligonucleotide having a nucleobase sequencecomplementary to miR-122. In certain embodiments, an anti-miR-122 isfully complementary to miR-122 (i.e., 100% complementary). In certainembodiments, an anti-miR-122 is at least 90%, at least 93%, at least94%, at least 95%, or 100% complementary. In certain embodiments, ananti-miR-122 is a modified oligonucleotide.

“Target nucleic acid” means a nucleic acid to which an oligomericcompound is designed to hybridize.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid.

“Modulation” means a perturbation of function, amount, or activity. Incertain embodiments, modulation means an increase in function, amount,or activity. In certain embodiments, modulation means a decrease infunction, amount, or activity.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“5′ target site” means the nucleobase of a target nucleic acid which iscomplementary to the 3′-most nucleobase of a particular oligonucleotide.

“3′ target site” means the nucleobase of a target nucleic acid which iscomplementary to the 5′-most nucleobase of a particular oligonucleotide.

“Region” means a portion of linked nucleosides within a nucleic acid. Incertain embodiments, an oligonucleotide has a nucleobase sequence thatis complementary to a region of a target nucleic acid. For example, incertain such embodiments an oligonucleotide is complementary to a regionof a microRNA sequence. In certain such embodiments, an oligonucleotideis fully complementary to a region of a microRNA.

“Segment” means a smaller or sub-portion of a region.

“Nucleobase sequence” means the order of contiguous nucleobases in anoligomeric compound or nucleic acid, typically listed in a 5′ to 3′orientation, independent of any sugar, linkage, and/or nucleobasemodification.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases topair non-covalently via hydrogen bonding.

“Complementary” means that one nucleic acid is capable of hybridizing toanother nucleic acid or oligonucleotide. In certain embodiments,complementary refers to an oligonucleotide capable of hybridizing to atarget nucleic acid.

“Fully complementary” means each nucleobase of an oligonucleotide iscapable of pairing with a nucleobase at each corresponding position in atarget nucleic acid. In certain embodiments, an oligonucleotide is fullycomplementary to a microRNA, i.e. each nucleobase of the oligonucleotideis complementary to a nucleobase at a corresponding position in themicroRNA. In certain embodiments, an oligonucleotide wherein eachnucleobase has complementarity to a nucleobase within a region of amicroRNA sequence is fully complementary to the microRNA sequence.

“Percent complementarity” means the percentage of nucleobases of anoligonucleotide that are complementary to an equal-length portion of atarget nucleic acid. Percent complementarity is calculated by dividingthe number of nucleobases of the oligonucleotide that are complementaryto nucleobases at corresponding positions in the target nucleic acid bythe total number of nucleobases in the oligonucleotide.

“Percent identity” means the number of nucleobases in a first nucleicacid that are identical to nucleobases at corresponding positions in asecond nucleic acid, divided by the total number of nucleobases in thefirst nucleic acid. In certain embodiments, the first nucleic acid is amicroRNA and the second nucleic acid is a microRNA. In certainembodiments, the first nucleic acid is an oligonucleotide and the secondnucleic acid is an oligonucleotide.

“Hybridize” means the annealing of complementary nucleic acids thatoccurs through nucleobase complementarity.

“Mismatch” means a nucleobase of a first nucleic acid that is notcapable of Watson-Crick pairing with a nucleobase at a correspondingposition of a second nucleic acid.

“Identical” in the context of nucleobase sequences, means having thesame nucleobase sequence, independent of sugar, linkage, and/ornucleobase modifications and independent of the methyl state of anypyrimidines present.

“MicroRNA” means an endogenous non-coding RNA between 18 and 25nucleobases in length, which is the product of cleavage of apre-microRNA by the enzyme Dicer. Examples of mature microRNAs are foundin the microRNA database known as miRBase(http://microma.sanger.ac.uk/). In certain embodiments, microRNA isabbreviated as “microRNA” or “miR.”

“Pre-microRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which is the product of cleavage of a pri-miR by thedouble-stranded RNA-specific ribonuclease known as Drosha.

“Stem-loop sequence” means an RNA having a hairpin structure andcontaining a mature microRNA sequence. Pre-microRNA sequences andstem-loop sequences may overlap. Examples of stem-loop sequences arefound in the microRNA database known as miRBase(http://microrna.sanger.ac.uk/).

“Pri-microRNA” or “pri-miR” means a non-coding RNA having a hairpinstructure that is a substrate for the double-stranded RNA-specificribonuclease Drosha.

“microRNA precursor” means a transcript that originates from a genomicDNA and that comprises a non-coding, structured RNA comprising one ormore microRNA sequences. For example, in certain embodiments a microRNAprecursor is a pre-microRNA. In certain embodiments, a microRNAprecursor is a pri-microRNA.

“microRNA-regulated transcript” means a transcript that is regulated bya microRNA. “Monocistronic transcript” means a microRNA precursorcontaining a single microRNA sequence.

“Polycistronic transcript” means a microRNA precursor containing two ormore microRNA sequences.

“Seed sequence” means a nucleobase sequence comprising from 6 to 8contiguous nucleobases of nucleobases 1 to 9 of the 5′-end of a maturemicroRNA sequence.

“Seed match sequence” means a nucleobase sequence that is complementaryto a seed sequence, and is the same length as the seed sequence.

“Oligomeric compound” means a compound that comprises a plurality oflinked monomeric subunits. Oligomeric compounds includedoligonucleotides.

“Oligonucleotide” means a compound comprising a plurality of linkednucleosides, each of which can be modified or unmodified, independentfrom one another.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalentlypairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar moiety.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of a nucleoside.

“Compound comprising a modified oligonucleotide consisting of a numberof linked nucleosides means a compound that includes a modifiedoligonucleotide having the specified number of linked nucleosides. Thus,the compound may include additional substituents or conjugates. Unlessotherwise indicated, the compound does not include any additionalnucleosides beyond those of the modified oligonucleotide.

“Modified oligonucleotide” means an oligonucleotide having one or moremodifications relative to a naturally occurring terminus, sugar,nucleobase, and/or internucleoside linkage. A modified oligonucleotidemay comprise unmodified nucleosides.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Modified nucleoside” means a nucleoside having any change from anaturally occurring nucleoside. A modified nucleoside may have amodified sugar, and an unmodified nucleobase. A modified nucleoside mayhave a modified sugar and a modified nucleobase. A modified nucleosidemay have a natural sugar and a modified nucleobase. In certainembodiments, a modified nucleoside is a bicyclic nucleoside. In certainembodiments, a modified nucleoside is a non-bicyclic nucleoside.

“2′-modified nucleoside” means a nucleoside comprising a sugar with anymodification at the position equivalent to the 2′ position of thefuranosyl ring as the positions are numbered in 2-deoxyribose or ribose.It is to be understood that 2′-modified nucleosides include, withoutlimitation, nucleosides comprising bicyclic sugar moieties.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom. A“phosphorothioate linkage” means a linkage between two chemical moietieshaving the same structure as a phosphorothioate internucleoside linkage,e.g., —OP(O)(S)O—.

A “phosphodiester linkage” means a linkage between two chemical moietieshaving the same structure as a phosphodiester internucleoside linkage,e.g., —OP(O)₂O—.

“Unmodified nucleobase” means the naturally occurring heterocyclic basesof RNA or DNA: the purine bases adenine (A) and guanine (G), and thepyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine),and uracil (U).

“5-methylcytosine” means a cytosine comprising a methyl group attachedto the 5 position.

“Non-methylated cytosine” means a cytosine that does not have a methylgroup attached to the 5 position.

“Modified nucleobase” means any nucleobase that is not an unmodifiednucleobase.

“Furanosyl” means a structure comprising a 5-membered ring consisting offour carbon atoms and one oxygen atom.

“Naturally occurring furanosyl” means a ribofuranosyl as found innaturally occurring RNA or a deoxyribofuranosyl as found in naturallyoccurring DNA.

“Sugar moiety” means a naturally occurring furanosyl or a modified sugarmoiety.

“Modified sugar moiety” means a substituted sugar moiety or a sugarsurrogate.

“Substituted sugar moiety” means a furanosyl that is not a naturallyoccurring furanosyl. Substituted sugar moieties include, but are notlimited to sugar moieties comprising modifications at the 2′-position,the 5′-position and/or the 4′-position of a naturally occurringfuranosyl. Certain substituted sugar moieties are bicyclic sugarmoieties.

“Sugar surrogate” means a structure that does not comprise a furanosyland that is capable of replacing the naturally occurring furanosyl of anucleoside, such that the resulting nucleoside is capable of (1)incorporation into an oligonucleotide and (2) hybridization to acomplementary nucleoside. Such structures include relatively simplechanges to the furanosyl, such as rings comprising a different number ofatoms (e.g., 4, 6, or 7-membered rings); replacement of the oxygen ofthe furanosyl with a non-oxygen atom (e.g., carbon, sulfur, ornitrogen); or both a change in the number of atoms and a replacement ofthe oxygen. Such structures may also comprise substitutionscorresponding with those described for substituted sugar moieties (e.g.,6-membered carbocyclic bicyclic sugar surrogates optionally comprisingadditional substituents). Sugar surrogates also include more complexsugar replacements (e.g., the non-ring systems of peptide nucleic acid).Sugar surrogates include without limitation morpholinos, cyclohexenylsand cyclohexitols.

“β-D-deoxyribose” means a naturally occurring DNA sugar moiety.

“β-D-ribose” means a naturally occurring RNA sugar moiety.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having a O-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having aO-methoxyethyl modification at the 2′ position.

“2′-O-fluoro” or “2′-F” means a sugar having a fluoro modification ofthe 2′ position.

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to7 membered ring (including by not limited to a furanosyl) comprising abridge connecting two atoms of the 4 to 7 membered ring to form a secondring, resulting in a bicyclic structure. In certain embodiments, the 4to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7membered ring is a furanosyl. In certain such embodiments, the bridgeconnects the 2′-carbon and the 4′-carbon of the furanosyl. Nonlimitingexemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, andR-cEt.

“Locked nucleic acid (LNA) sugar moiety” means a substituted sugarmoiety comprising a (CH₂)—O bridge between the 4′ and 2′ furanose ringatoms.

“ENA sugar moiety” means a substituted sugar moiety comprising a(CH₂)₂—O bridge between the 4′ and 2′ furanose ring atoms.

“Constrained ethyl (cEt) sugar moiety” means a substituted sugar moietycomprising a CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms. In certain embodiments, the CH(CH₃)—O bridge is constrained inthe S orientation. In certain embodiments, the CH(CH₃)—O bridge isconstrained in the R orientation.

“S-cEt sugar moiety” means a substituted sugar moiety comprising anS-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“R-cEt sugar moiety” means a substituted sugar moiety comprising anR-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“2′-O-methyl nucleoside” means a modified nucleoside having a2′-O-methyl sugar modification.

“2′-O-methoxyethyl nucleoside” means a modified nucleoside having a2′-O-methoxyethyl sugar modification. A 2′-O-methoxyethyl nucleoside maycomprise a modified or unmodified nucleobase.

“2′-fluoro nucleoside” means a modified nucleoside having a 2′-fluorosugar modification. A 2′-fluoro nucleoside may comprise a modified orunmodified nucleobase.

“Bicyclic nucleoside” means a modified nucleoside having a bicyclicsugar moiety. A bicyclic nucleoside may have a modified or unmodifiednucleobase.

“cEt nucleoside” means a nucleoside comprising a cEt sugar moiety. A cEtnucleoside may comprise a modified or unmodified nucleobase.

“S-cEt nucleoside” means a nucleoside comprising an S-cEt sugar moiety.

“R-cEt nucleoside” means a nucleoside comprising an R-cEt sugar moiety.

“Non-bicyclic nucleoside” means a nucleoside that has a sugar other thana bicyclic sugar. In certain embodiments, a non-bicyclic nucleosidecomprises a naturally occurring sugar. In certain embodiments, anon-bicyclic nucleoside comprises a modified sugar. In certainembodiments, a non-bicyclic nucleoside is a β-D-deoxyribonucleoside. Incertain embodiments, a non-bicyclic nucleoside is a 2′-O-methoxyethylnucleoside.

“β-D-deoxyribonucleoside” means a naturally occurring DNA nucleoside.

“β-D-ribonucleoside” means a naturally occurring RNA nucleoside.

“LNA nucleoside” means a nucleoside comprising a LNA sugar moiety.

“ENA nucleoside” means a nucleoside comprising an ENA sugar moiety.

“Motif′ means a pattern of modified and/or unmodified nucleobases,sugars, and/or internucleoside linkages in an oligonucleotide. Incertain embodiments, a motif is a nucleoside pattern.

“Nucleoside pattern” means a pattern of nucleoside modifications in amodified oligonucleotide or a region thereof. A nucleoside pattern is amotif that describes the arrangement of nucleoside modifications in anoligonucleotide.

“Fully modified oligonucleotide” means each nucleobase, each sugar,and/or each internucleoside linkage is modified.

“Uniformly modified oligonucleotide” means each nucleobase, each sugar,and/or each internucleoside linkage has the same modification throughoutthe modified oligonucleotide.

“Stabilizing modification” means a modification to a nucleoside thatprovides enhanced stability to a modified oligonucleotide, in thepresence of nucleases, relative to that provided by 2′-deoxynucleosideslinked by phosphodiester internucleoside linkages. For example, incertain embodiments, a stabilizing modification is a stabilizingnucleoside modification. In certain embodiments, a stabilizingmodification is an internucleoside linkage modification.

“Stabilizing nucleoside” means a nucleoside modified to provide enhancednuclease stability to an oligonucleotide, relative to that provided by a2′-deoxynucleoside. In one embodiment, a stabilizing nucleoside is a2′-modified nucleoside.

“Stabilizing internucleoside linkage” means an internucleoside linkagethat provides improved nuclease stability to an oligonucleotide relativeto that provided by a phosphodiester internucleoside linkage. In oneembodiment, a stabilizing internucleoside linkage is a phosphorothioateinternucleoside linkage.

A “linking group” as used herein refers to an atom or group of atomsthat attach a first chemical entity to a second chemical entity via oneor more covalent bonds.

A “linker” as used herein, refers to an atom or group of atoms thatattach one or more ligands to a modified or unmodified nucleoside viaone or more covalent bonds. The modified or unmodified nucleoside may bepart of a modified oligonucleotide as described herein, or may beattached to a modified oligonucleotide through a phosphodiester orphosphorothioate bond. In some embodiments, the linker attaches one ormore ligands to the 3′ end of a modified oligonucleotide. In someembodiments, the linker attaches one or more ligands to the 5′ end of amodified oligonucleotide. In some embodiments, the linker attaches oneor more ligands to a modified or unmodified nucleoside that is attachedto the 3′ end of a modified oligonucleotide. In some embodiments, thelinker attaches one or more ligands to a modified or unmodifiednucleoside that is attached to the 5′ end of a modified oligonucleotide.When the linker attaches one or more ligands to the 3′ end of a modifiedoligonucleotide or to a modified or unmodified nucleoside attached tothe 3′ end of a modified oligonucleotide, in some embodiments, theattachment point for the linker may be the 3′ carbon of a modified orunmodified sugar moiety. When the linker attaches one or more ligands tothe 5′ end of a modified oligonucleotide or to a modified or unmodifiednucleoside attached to the 5′ end of a modified oligonucleotide, in someembodiments, the attachment point for the linker may be the 5′ carbon ofa modified or unmodified sugar moiety.

Overview

To identify potent inhibitors of miR-122, numerous anti-miR-122compounds were designed and synthesized. The compounds comprisedmodified oligonucleotides that varied in length, and in the number,placement, and identity of bicyclic nucleosides and non-bicyclicnucleosides. An initial series of compounds was tested in an in vitroluciferase assay, which identified a subset of compounds as in vitroactive compounds. These in vitro active compounds were then tested in invivo assays to identify those compounds that are potent inhibitors ofmiR-122 in vivo. From the initial in vitro and in vivo screens, certaincompounds were selected as the basis for the design of additionalcompounds. The experimentally observed correlations between structureand activity (both in vitro and in vivo) were used to inform the designof these additional compounds, with further variations in length andselection and arrangement of bicyclic and non-bicyclic nucleosides. Thein vitro and in vivo screening assays were repeated for these additionalcompounds. Certain compounds were also tested for other properties, forexample, susceptibility to exonuclease activity, tissue accumulation,and tissue half-life.

Of over 400 compounds screened in vitro during this process,approximately 150 were identified as active in an in vitro luciferaseassay. Approximately 70 of these compounds were further evaluated for invivo potency and safety. Through this iterative process of designing andscreening compounds, it was observed that certain compounds, bothunconjugated anti-miR-122 modified oligonucleotides and conjugatedanti-miR-122 modified oligonucleotides, were potent inhibitors ofmiR-122 in vivo. As such, these compounds are useful for the modulationof cellular processes that are promoted by the activity of miR-122.Further, such compounds are useful for treating, preventing, and/ordelaying the onset of diseases associated with miR-122. Such diseasesinclude, but are not limited to, HCV infection and HCV-relatedcomplications, such as cirrhosis, liver fibrosis, steatohepatitis,steatosis, and hepatocellular carcinoma.

Certain Anti-miR-122 Compounds

Provided herein are modified oligonucleotides having certain patterns ofbicyclic and non-bicyclic nucleosides. Modified oligonucleotides havingthe patterns identified herein are effective inhibitors of miR-122activity.

Each of the nucleoside patterns illustrated herein is shown in the 5′ to3′ orientation.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of from 16 to 22 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-122 (SEQ ID NO: 1) and wherein the modifiedoligonucleotide comprises at least 16 contiguous nucleosides of thefollowing nucleoside pattern I in the 5′ to 3′ orientation:

(R)_(X)-N^(Q)-N^(Q)-N^(B)-N^(B)-N^(Q)-N^(B)-N^(Q)-N^(B)-N^(Q)-N^(B)-N^(B)-(N^(Z))_(Y)

wherein each R is, independently, a non-bicyclic nucleoside or abicyclic nucleoside;

X is from 4 to 10;

each N^(B) is, independently, a bicyclic nucleoside;

each N^(Q) is, independently, a non-bicyclic nucleoside;

Y is 0 or 1; and

N^(Z) is a modified nucleoside or an unmodified nucleoside non-bicyclicnucleoside or a bicyclic nucleoside.

In certain embodiments, the modified oligonucleotide comprises at least16, at least 17, at least 18, at least 19, at least 20, at least 21, or22 contiguous nucleosides of nucleoside pattern I.

In certain embodiments, each bicyclic nucleoside is independentlyselected from an LNA nucleoside, a cEt nucleoside, and an ENAnucleoside.

In certain embodiments, at least two bicyclic nucleosides are differentfrom one another.

In certain embodiments, all bicyclic nucleosides have the same type ofsugar moiety.

In certain embodiments, each bicyclic nucleoside is a cEt nucleoside. Incertain embodiments, the cEt nucleoside is an S-cEt nucleoside. Incertain embodiments, the cEt nucleoside is an R-cEt nucleoside.

In certain embodiments, each bicyclic nucleoside is an LNA nucleoside.

In certain embodiments, at least two non-bicyclic nucleosides comprisesugar moieties that are different from one another. In certainembodiments, each non-bicyclic nucleoside has the same type of sugarmoiety.

In certain embodiments, each non-bicyclic nucleoside is independentlyselected from a β-D-deoxyribonucleoside, a β-D-ribonucleoside,2′-O-methyl nucleoside, a 2′-O-methoxyethyl nucleoside, and a2′-fluoronucleoside. In certain embodiments, each non-bicyclicnucleoside is independently selected from a β-D-deoxyribonucleoside, anda 2′-O-methoxyethyl nucleoside. In certain embodiments, eachnon-bicyclic nucleoside is a β-D-deoxyribonucleoside. In certainembodiments, each non-bicyclic nucleoside is a 2′-MOE nucleoside.

In certain embodiments, no more than two non-bicyclic nucleosides are2′-MOE nucleosides. In certain embodiments, no more than twonon-bicyclic nucleosides are 2′-MOE nucleosides, and each othernon-bicyclic nucleoside is a β-D-deoxyribonucleoside.

In certain embodiments, the 5′-terminal and the 3′-terminal non-bicyclicnucleosides are 2′-MOE nucleosides and each other non-bicyclicnucleoside is a β-D-deoxyribonucleoside.

In certain embodiments, two non-bicyclic nucleosides are 2′-MOEnucleosides and each other non-bicyclic nucleoside is aβ-D-deoxyribonucleoside.

In certain embodiments, each nucleoside of R is a 2′-MOE nucleoside.

In certain embodiments, X is 4, 5, 6, 7, 8, 9, or 10.

In certain embodiments, Y is 0. In certain embodiments, Y is 1.

In certain embodiments, R consist of seven linked nucleosides, whereineach nucleoside is a 2′-O-methoxyethyl nucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, R consists of four linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4) wherein each of N^(R1) and N^(R3) is a S-cEtnucleoside and each of N^(R2) and N^(R4) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; Y is 1; and N^(Z) is a β-D-deoxyribonucleoside.

In certain embodiments, R consists of four linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4) wherein each of N^(R1) and N^(R4) is a S-cEtnucleoside and each of N^(R2) and N^(R3) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; Y is 1; and N^(Z) is a 2′-O-methoxyethylnucleoside.

In certain embodiments, R consists of seven linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), and N^(R4) is a 2′-O-methoxyethyl nucleoside,each of N^(R5) and N^(R7) is a β-D-deoxyribonucleoside, and N^(R6) isS-cEt nucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, R consists of seven linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), N^(R4), and N^(R5) is a 2′-O-methoxyethylnucleoside, N^(R6) is S-cEt nucleoside, and N^(R7) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, R consists of seven linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is 2′-O-methoxyethylnucleoside, and N^(R7) is a β-D-deoxyribonucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, R consists of ten linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7)-N^(R8)-N^(R9)-N^(R10),wherein each of N^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is2′-O-methoxyethyl nucleoside, each of N^(R7) and N^(R9) is a an S-cEtnucleoside; each of N^(R8) and N^(R10) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and Y is 0.

In certain embodiments, R consists of ten linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7)-N^(R8)-N^(R9)-N^(R10),wherein each of N^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is2′-O-methoxyethyl nucleoside, each of N^(R7) and N^(R9) is a an S-cEtnucleoside; and each of N^(R8) and N^(R10) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; Y is 1 and N_(z) is a 2′-O-methoxyethylnucleoside.

In certain embodiments, R consists of four linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4) wherein each of N^(R1) and N^(R4) is anS-cEt nucleoside, and each of N^(R1) and N^(R3) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; Y is 1 and N^(Z) is aβ-D-deoxyribonucleoside.

In certain embodiments, R consists of four linked nucleosidesN^(R1)-N^(R2)-N^(R3)-N^(R4) wherein N^(R1) is a 2′-O-methoxyethylnucleoside, each of N^(R2) and N^(R4) is an S-cEt nucleoside, and N^(R3)is a β-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; Y is 1 and N^(Z) is a2′-O-methoxyethyl nucleoside.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 90%, at least 93%, at least 94%, at least95%, or 100% complementary to the nucleobase sequence of miR-122 (SEQ IDNO: 1).

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is complementary to miR-122 such that position 2 of SEQID NO: 1 is paired with the 3′-terminal nucleobase of theoligonucleotide. For example:

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is complementary to miR-122 such that position 1 of SEQID NO: 1 is paired with the 3′-terminal nucleobase of theoligonucleotide. For example:

In certain embodiments, at least one internucleoside linkage is amodified internucleoside linkage. In certain embodiments, eachinternucleoside linkage is a modified internucleoside linkage. Incertain embodiments, a modified internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, at least one pyrimidine of the modifiedoligonucleotide comprises a 5-methyl group. In certain embodiments, atleast one cytosine of the modified oligonucleotide is a5-methylcytosine. In certain embodiments, each cytosine of the modifiedoligonucleotide is a 5-methylcytosine. In certain embodiments, eachmodified nucleotide that comprises a cytosine comprises a5-methylcytosine. In certain embodiments, each2′-O-methoxyethylnucleoside that comprises a cytosine comprises a5-methylcytosine.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is selected from SEQ ID NOs: 3 to 6, wherein each T isindependently selected from T and U.

In certain embodiments, the modified oligonucleotide has 0, 1, 2, or 3mismatches with respect to the nucleobase sequence of miR-122. Incertain embodiments, the modified oligonucleotide has 0 mismatches withrespect to the nucleobase sequence of miR-122. In certain embodiments,the modified oligonucleotide has 1 mismatch with respect to thenucleobase sequence of miR-122. In certain embodiments, the modifiedoligonucleotide has 2 mismatches with respect to the nucleobase sequenceof miR-122.

In certain embodiments, a modified oligonucleotide consists of greaterthan 22 linked nucleosides, and comprises at least 8 linked nucleosidesof nucleoside pattern I. The nucleosides that are present in addition tothe nucleosides described by nucleoside pattern I are either modified orunmodified.

In certain embodiments, a modified oligonucleotide consists of less than16 linked nucleosides, and comprises at least 8 linked nucleosides ofnucleoside pattern I.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence and modifications as shown in Table 1. Nucleosides andnucleobases are indicated as follows: the superscript “Me” indicates5-methylcytosine; nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are2′-MOE nucleosides; nucleosides followed by a subscript “S” are S-cEtnucleosides; and each internucleoside linkage is a phosphorothioateinternucleoside linkage.

TABLE 1 Anti-miR-122 Compounds Com- SEQ pound ID # Sequence andModifications NO 38649 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 38012C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 38016^(Me)C_(S)CAT_(S)TGT_(S) ^(Me)C_(S)A^(Me)C_(S)A^(Me)C_(S)T^(Me)C_(S)^(Me)C_(S)A_(E) 3 38646 A_(E) ^(Me)C_(E)A_(E)^(Me)C_(E)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 38647 A_(E)^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 38648 A_(E)^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 38652^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 5 38659C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E) 10 38660^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E) 638872 C_(S)CAU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 38910^(Me)C_(E)C_(S)AU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A_(E) 3

In some embodiments, a modified oligonucleotide has a nucleobasesequence and modifications as shown below:

U_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A_(S) (SEQ ID NO: 8); or

C_(S)A_(S)C_(S)A_(S)C_(S)U_(S)C_(S)C_(S) (SEQ ID NO: 9);

wherein nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “S” areS-cEt nucleosides; and each internucleoside linkage is aphosphorothioate internucleoside linkage. In some such embodiments, acompound is 38591, 38633, 38998, or 38634.

Anti-miR-122 Compounds Comprising Conjugates

In certain embodiments, a compound provided herein comprises a modifiedoligonucleotide conjugated to one or more moieties which enhance theactivity, cellular distribution and/or cellular uptake of theoligonucleotide. For example, increased cellular uptake of compounds maybe achieved by utilizing conjugates that are ligands for cell-surfacereceptors. The binding of a ligand conjugated to an exogenous molecule(e.g., a drug) to its cell surface receptor leads to the internalizationof the conjugated molecule, thereby enhancing transmembrane transport ofthe exogenous molecule. Any of the anti-miR-122 modifiedoligonucleotides provided herein may be linked to one or more moietiesto form a compound comprising a conjugated anti-miR-122 modifiedoligonucleotide.

In certain embodiments, a compound provided herein comprises a conjugatemoiety linked to the 5′ terminus or the 3′ terminus of the modifiedoligonucleotide. In certain embodiments, the compound comprises aconjugate moiety linked to the 3′ terminus of the modifiedoligonucleotide. In certain embodiments, the compound comprises aconjugate moiety linked to the 5′ terminus of the modifiedoligonucleotide. In certain embodiments, the compound comprises a firstconjugate moiety linked to the 3′ terminus of the modifiedoligonucleotide and a second conjugate moiety linked to the 5′ terminusof the modified oligonucleotide.

In certain embodiments, a conjugate moiety comprises at least one ligandselected from a carbohydrate, cholesterol, a lipid, a phospholipid, anantibody, a lipoprotein, a hormone, a peptide, a vitamin, a steroid, ora cationic lipid.

Ligands may be covalently attached to a modified oligonucleotide by anysuitable linker. Various linkers are known in the art, and certainnonlimiting exemplary linkers are described, e.g., in PCT PublicationNo. WO 2013/033230 and U.S. Pat. No. 8,106,022B2. In some embodiments, alinker may be selected that is resistant to enzymatic cleavage in vivo.In some embodiments, a linker may be selected that is resistant tohydrolytic cleavage in vivo. In some embodiments, a linker may beselected that will undergo enzymatic cleavage in vivo. In someembodiments, a linker may be selected that will undergo hydrolyticcleavage in vivo.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has the structure:

L-X₁-N_(m)-X₂-MO;

wherein each L is a ligand; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; X₁ and X₂ are each,independently, a phosphodiester linkage or a phosphorothioate linkage;and MO is a modified oligonucleotide. In certain embodiments, m is 1. Incertain embodiments, m is 2. In certain embodiments, m is 2, 3, 4, or 5.In certain embodiments, m is 3, 4, or 5. In certain embodiments, when mis greater than 1, each modified or unmodified nucleoside of N_(m) maybe connected to adjacent modified or unmodified nucleosides of N_(m) bya phosphodiester internucleoside linkage or a phosphorothioateinternucleoside linkage. In certain embodiments, m is 1 and X₁ and X₂are each phosphodiester.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure A:

L_(n)-linker-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; and MOis a modified oligonucleotide.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure B:

L_(n)-linker-X₁-N_(m)-X₂-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; each Nis, independently, a modified or unmodified nucleoside and m is from 1to 5; X₁ and X₂ are each, independently, a phosphodiester linkage or aphosphorothioate linkage; and MO is a modified oligonucleotide. Incertain embodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 2, 3, 4, or 5. In certain embodiments, m is 3, 4, or5. In certain embodiments, when m is greater than 1, each modified orunmodified nucleoside of N_(m) may be connected to adjacent modified orunmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure C:

L_(n)-linker-X-N_(m)-Y-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; each Nis, independently, a modified or unmodified nucleoside and m is from 1to 5; X is a phosphodiester linkage or a phosphorothioate linkage; Y isa phosphodiester linkage; and MO is a modified oligonucleotide. Incertain embodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 2, 3, 4, or 5. In certain embodiments, m is 3, 4, or5. In certain embodiments, when m is greater than 1, each modified orunmodified nucleoside of N_(m) may be connected to adjacent modified orunmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprising a conjugated modifiedoligonucleotide described herein has Structure D:

L_(n)-linker-Y-N_(m)-Y-MO;

wherein each L is, independently, a ligand and n is from 1 to 10; each Nis, independently, a modified or unmodified nucleoside and m is from 1to 5; each Y is a phosphodiester linkage; and MO is a modifiedoligonucleotide. In certain embodiments, m is 1. In some embodiments, mis 2. In certain embodiments, m is 3, 4, or 5. In certain embodiments, mis 2, 3, 4, or 5. In certain embodiments, when m is greater than 1, eachmodified or unmodified nucleoside of N_(m) may be connected to adjacentmodified or unmodified nucleosides of N_(m) by a phosphodiesterinternucleoside linkage or phosphorothioate internucleoside linkage.

In certain embodiments, when n is greater than 1, the linker comprises ascaffold capable of linking more than one L to the remainder of thecompound (i.e., to the modified oligonucleotide (MO), to X₁-N_(m)-X₂-MO,to X-N_(m)-Y-MO, etc.). In some such embodiments, the L_(n)-linkerportion of the compound (such as a compound of Structure A, B, C, or D)comprises Structure E:

L-Q′

S-Q″-

wherein each L is, independently, a ligand; n is from 1 to 10; S is ascaffold; and Q′ and Q″ are, independently, linking groups.

In certain embodiments, each Q′ and Q″ is independently selected from apeptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl, asubstituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

In certain embodiments, a scaffold links 2, 3, 4, or 5 ligands to amodified oligonucleotide. In certain embodiments, a scaffold links 3ligands to a modified oligonucleotide.

A nonlimiting exemplary Structure E is Structure E(i):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, and R₄are each, independently, selected from H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, and R₄ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments, R₁, R₂, R₃, and R₄ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(ii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁ is selected fromH, C₁-C₆ alkyl, and substituted C₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁ is selected from H, methyl, ethyl, propyl,isopropyl, and butyl. In some embodiments, R₁ is H or methyl.

A further nonlimiting exemplary Structure E is Structure E(iii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, R₄, andR₅ are each, independently, selected from H, C₁-C₆ alkyl, andsubstituted C₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, R₄, and R₅ are each,independently, selected from H, methyl, ethyl, propyl, isopropyl, andbutyl. In some embodiments R₁, R₂, R₃, R₄, and R₅ are each selected fromH and methyl.

A further nonlimiting exemplary Structure E is Structure E(iv):

wherein L₁ and L₂ are each, independently, a ligand; Q′₁, Q′₂, and Q″are each, independently, a linking group; and R₁, R₂, and R₃ are each,independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, and Q″ are each, independently, selectedfrom a peptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl,a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid. In someembodiments, R₁, R₂, and R₃ are each, independently, selected from H,methyl, ethyl, propyl, isopropyl, and butyl. In some embodiments R₁, R₂,and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(v):

wherein L₁ and L₂ are each, independently, a ligand; Q′₁, Q′₂, and Q″are each, independently, a linking group; and R₁, R₂, and R₃ are each,independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, and Q″ are each, independently, selectedfrom a peptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl,a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid. In someembodiments, R₁, R₂, and R₃ are each, independently, selected from H,methyl, ethyl, propyl, isopropyl, and butyl. In some embodiments R₁, R₂,and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(vi):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, and R₃ areeach, independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, and R₃ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, and R₃ are each selected from H and methyl.

A further nonlimiting exemplary Structure E is Structure E(vii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; R₁, R₂, and R₃ areeach, independently, selected from H, C₁-C₆ alkyl, and substituted C₁-C₆alkyl; and Z and Z′ are each independently selected from O and S.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, and R₃ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, and R₃ are each selected from H and methyl. In someembodiments, Z or Z′ on at least one P atom is S, and the other Z or Z′is O (i.e., a phosphorothioate linkage). In some embodiments, each—OP(Z)(Z′)O— is a phosphorothioate linkage. In some embodiments, Z andZ′ are both O on at least one P atom (i.e., a phosphodiester linkage).In some embodiments, each —OP(Z)(Z′)O— is a phosphodiester linkage.

A further nonlimiting exemplary Structure E is Structure E(viii):

wherein L₁, L₂, and L₃ are each, independently, a ligand; Q′₁, Q′₂, Q′₃,and Q″ are each, independently, a linking group; and R₁, R₂, R₃, and R₄are each, independently, selected from H, C₁-C₆ alkyl, and substitutedC₁-C₆ alkyl.

In some embodiments, Q′₁, Q′₂, Q′₃, and Q″ are each, independently,selected from a peptide, an ether, polyethylene glycol, an alkyl, aC₁-C₂₀ alkyl, a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, asubstituted C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀alkynyl, a C₁-C₂₀ alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, apyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and 6-aminohexanoicacid. In some embodiments, R₁, R₂, R₃, and R₄ are each, independently,selected from H, methyl, ethyl, propyl, isopropyl, and butyl. In someembodiments R₁, R₂, R₃, and R₄ are each selected from H and methyl.

Nonlimiting exemplary scaffolds and/or linkers comprising scaffolds, andsynthesis thereof, are described, e.g., PCT Publication No. WO2013/033230, U.S. Pat. No. 8,106,022B2, U.S. Publication No.2012/0157509 A1; U.S. Pat. No. 5,994,517; U.S. Pat. No. 7,491,805B2;U.S. Pat. No. 8,313,772B2; Manoharan, M., Chapter 16, Antisense DrugTechnology, Crooke, S.T., Marcel Dekker, Inc., 2001, 391-469.

In certain embodiments, the L_(n)-linker portion of the compoundcomprises Structure F:

wherein:

B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,—Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—;

MO is a modified oligonucleotide;

R^(N) is selected from H, methyl, ethyl, propyl, isopropyl, butyl, andbenzyl;

Z, Z′, and Z″ are each independently selected from 0 and S;

each N is, independently, a modified or unmodified nucleoside;

m is from 1 to 5;

X is selected from a phosphodiester linkage and a phosphorothioatelinkage;

Y is a phosphodiester linkage; and

the wavy line indicates the connection to the rest of the linker andligand(s).

In certain embodiments, the wavy line indicates a connection toStructure E, above.

In certain embodiments, n is from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Incertain embodiments, n is 1.

In certain embodiments, n is 2. In certain embodiments, n is 3. Incertain embodiments, n is 4. In certain embodiments, n is 5.

In certain embodiments, the L_(n)-linker portion of the compoundcomprises Structure G:

wherein:B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,—Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—;

MO is a modified oligonucleotide;

R^(N) is selected from H, methyl, ethyl, propyl, isopropyl, butyl, andbenzyl;

Z, Z′, and Z″ are each independently selected from 0 and S;

each N is, independently, a modified or unmodified nucleoside;

m is from 1 to 5;

X is selected from a phosphodiester linkage and a phosphorothioatelinkage;

Y is a phosphodiester linkage;

each L is, independently, a ligand; n is from 1 to 10; S is a scaffold;and Q′ and Q″ are, independently, linking groups.

In certain embodiments, each Q′ and Q″ are independently selected from apeptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl, asubstituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.

A nonlimiting exemplary L_(n)-linker portion (e.g., of Structure F or G)of a compound is shown in

Structure H below:

wherein the wavy line indicates attachment to the modifiedoligonucleotide (MO), to X₁, e.g. in Structure B, or to X or Y, e.g., inStructure C, or D.

In certain embodiments, each ligand is a carbohydrate. A compoundcomprising a carbohydrate-conjugated modified oligonucleotide, whenrecognized by a cell surface lectin, is transported across the cellmembrane into the cell. In certain embodiments, a cell surface lectin isa C-type lectin. In certain embodiments, the C-type lectin is present ona Kuppfer cell. In certain embodiments, a C-type lectin is present on amacrophage. In certain embodiments, a C-type lectin is present on anendothelial cell. In certain embodiments, a C-type lectin is present ona monocyte. In certain embodiments, a C-type lectin is present on aleukocyte. In certain embodiments, a C-type lectin is present on adendritic cell. In certain embodiments, a C-type lectin is present on aB cell. A conjugate may facilitate uptake of an anti-miR-122 compoundinto any cell type that expresses a C-type lectin.

In certain embodiments, a C-type lectin is the asialoglycoproteinreceptor (ASGPR). In certain embodiments, a conjugate comprises one ormore ligands having affinity for the ASGPR, including but not limited togalactose or a galactose derivative. In certain embodiments, a ligandhaving affinity for the ASGPR is N-acetylgalactosamine, galactose,galactosamine, N-formylgalactosamine, N-propionyl-galactosamine,N-n-butanoylgalactosamine, or N-iso-butanoyl-galactosamine. Suchconjugates facilitate the uptake of compounds into cells that expressthe ASGPR, for example, hepatocytes and dendritic cells.

In certain embodiments, a ligand is a carbohydrate selected frommannose, glucose, galactose, ribose, arabinose, fructose, fucose,xylose, D-mannose, L-mannose, D-galactose, L-galactose, D-glucose,L-glucose, D-ribose, L-ribose, D-arabinose, L-arabinose, D-fructose,L-fructose, D-fucose, L-fucose, D-xylose, L-xylose,alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose,beta-D-mannopyranose, alpha-D-glucofuranose, Beta-D-glucofuranose,alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-galactofuranose,beta-D-galactofuranose, alpha-D-galactopyranose, beta-D-galactopyranose,alpha-D-ribofuranose, beta-D-ribofuranose, alpha-D-ribopyranose,beta-D-ribopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose,glucosamine, galactosamine, sialic acid, and N-acetylgalactosamine.

In certain embodiments, a ligand is selected from N-acetylgalactosamine,galactose, galactosamine, N-formylgalactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, andN-iso-butanoyl-galactosamine

In certain embodiments, a ligand is N-acetylgalactosamine.

In certain embodiments, a compound comprises the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide. In certain embodiments, m is 1. In certainembodiments, m is 2. In certain embodiments, m is 3, 4, or 5. In certainembodiments, m is 2, 3, 4, or 5. In certain embodiments, when m isgreater than 1, each modified or unmodified nucleoside of N_(m) may beconnected to adjacent modified or unmodified nucleosides of N_(m) by aphosphodiester internucleoside linkage or phosphorothioateinternucleoside linkage.

In certain embodiments, a compound comprises the structure:

wherein X is a phosphodiester linkage or a phosphorothioate linkage;each N is, independently, a modified or unmodified nucleoside and m isfrom 1 to 5; Y is a phosphodiester linkage; and MO is a modifiedoligonucleotide. In certain embodiments, m is 1. In certain embodiments,m is 2. In certain embodiments, m is 2, 3, 4, or 5. In certainembodiments, m is 3, 4, or 5. In certain embodiments, when m is greaterthan 1, each modified or unmodified nucleoside of N_(m) may be connectedto adjacent modified or unmodified nucleosides of N_(m) by aphosphodiester internucleoside linkage or phosphorothioateinternucleoside linkage.

In certain embodiments, a compound comprises a modified nucleotide and aconjugate moiety, wherein the modified oligonucleotide has the structureC_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) (SEQ ID NO: 7), whereinthe subscript “L” indicates an LNA and nucleosides not followed by asubscript are β-D-deoxyribonucleosides, and each internucleoside linkageis a phosphorothioate internucleoside linkage, and wherein the conjugatemoiety is linked to the 3′ terminus of the modified oligonucleotide andhas the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide. In some embodiments, all of the C_(L)nucleosides are ^(Me)C_(L) nucleosides, wherein the superscript “Me”indicates 5-methylcytosine.

In some embodiments, a compound has the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide.

In certain embodiments, at least one of X₁ and X₂ is a phosphodiesterlinkage. In certain embodiments, each of X₁ and X₂ is a phosphodiesterlinkage.

In certain embodiments, m is 1. In certain embodiments, m is 2. Incertain embodiments, m is 3, 4, or 5. In certain embodiments, m is 2, 3,4, or 5. In certain embodiments, when m is greater than 1, each modifiedor unmodified nucleoside of N_(m) may be connected to adjacent modifiedor unmodified nucleosides of N_(m) by a phosphodiester internucleosidelinkage or a phosphorothioate internucleoside linkage.

In any of the embodiments described herein, N_(m) may be N′_(p)N“, whereeach N′ is, independently, a modified or unmodified nucleoside and p isfrom 0 to 4; and N” is a nucleoside comprising an unmodified sugarmoiety.

In certain embodiments, p is 0. In certain embodiments, p is 1, 2, 3, or4. In certain embodiments, when p is 1, 2, 3, or 4, each N′ comprises anunmodified sugar moiety.

In certain embodiments, an unmodified sugar moiety is a β-D-ribose or aβ-D-deoxyribose. In certain embodiments, where p is 1, 2, 3, or 4, N′comprises a purine nucleobase. In certain embodiments, N″ comprises apurine nucleobase. In certain embodiments, a purine nucleobase isselected from adenine, guanine, hypoxanthine, xanthine, and7-methylguanine. In certain embodiments, N′ is a 13-D-deoxyriboadenosineor a β-D-deoxyriboguanosine. In certain embodiments, N″ is aβ-D-deoxyriboadenosine or a β-D-deoxyriboguanosine.

In certain embodiments, p is 1, N′ and N″ are each aβ-D-deoxyriboadenosine, and N′ and N″ are linked by a phosphodiesterinternucleoside linkage. In certain embodiments, p is 1, N′ and N″ areeach a β-D-deoxyriboadenosine, and N′ and N″ are linked by aphosphodiester internucleoside linkage. In certain embodiments, p is 1,N′ and N″ are each a β-D-deoxyriboadenosine, and N′ and N″ are linked bya phosphorothioate internucleoside linkage.

In certain embodiments, where p is 1, 2, 3, or 4, N′ comprises apyrimidine nucleobase. In certain embodiments, N″ comprises a pyrimidinenucleobase. In certain embodiments, a pyrimidine nucleobase is selectedfrom cytosine, 5-methylcytosine, thymine, uracil, and 5,6-dihydrouracil.

In certain embodiments, the sugar moiety of each N is independentlyselected from a β-D-ribose, a β-D-deoxyribose, a 2′-O-methoxy sugar, a2′-O-methyl sugar, a 2′-fluoro sugar, and a bicyclic sugar moiety. Incertain embodiments, each bicyclic sugar moiety is independentlyselected from a cEt sugar moiety, an LNA sugar moiety, and an ENA sugarmoiety. In certain embodiments, the cEt sugar moiety is an S-cEt sugarmoiety. In certain embodiments, the cEt sugar moiety is an R-cEt sugarmoiety.

In certain embodiments, a compound comprises the structure:

wherein X is a phosphodiester linkage; m is 1; N is aβ-D-deoxyriboadenosine; Y is a phosphodiester linkage; and MO is amodified oligonucleotide.

In certain embodiments, a compound comprises the structure:

wherein X is a phosphodiester linkage; m is 2; each N is aβ-D-deoxyriboadenosine; the nucleosides of N are linked by aphosphodiester internucleoside linkage; Y is a phosphodiester linkage;and MO is a modified oligonucleotide.

In certain embodiments, a compound comprises a modified nucleotide and aconjugate moiety, wherein the modified oligonucleotide has the structureA_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) (SEQ ID NO: 4),where nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are2′-MOE nucleosides; nucleosides followed by a subscript “S” are S-cEtnucleosides; and each internucleoside linkage is a phosphorothioateinternucleoside linkage; and wherein the conjugate moiety is linked tothe 3′ terminus of the modified oligonucleotide and has the structure:

wherein X is a phosphodiester linkage; m is 1; N is aβ-D-deoxyriboadenosine; Y is a phosphodiester linkage; and MO is themodified oligonucleotide.

In certain embodiments, a compound comprises a modified nucleotide and aconjugate moiety, wherein the modified oligonucleotide has the structureC_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) (SEQ ID NO: 7), whereinthe subscript “L” indicates an LNA and nucleosides not followed by asubscript are β-D-deoxyribonucleosides, and each internucleoside linkageis a phosphorothioate internucleoside linkage, and wherein the conjugatemoiety is linked to the 3′ terminus of the modified oligonucleotide andhas the structure:

wherein X is a phosphodiester linkage; m is 1; N is aβ-D-deoxyriboadenosine; Y is a phosphodiester linkage; and MO is themodified oligonucleotide. In some embodiments, all of the C_(L)nucleosides are ^(Me)C_(L) nucleosides, wherein the superscript “Me”indicates 5-methylcytosine.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence and modifications as shown in Table 2. Nucleosides andnucleobases are indicated as follows: the superscript “Me” indicates5-methylcytosine; nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are2′-MOE nucleosides; nucleosides followed by a subscript “S” are S-cEtnucleosides; and each internucleoside linkage is a phosphorothioateinternucleoside linkage.

TABLE 2 Conjugated modified oligonucleotides Cmpd SEQ ID # Sequence (5′to 3′) and Modifications Linkage to GalNAc structure NO 38368 A_(E)^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s) C_(s) AC_(s) A C_(s) T C_(s) C_(s) Structure III of Figure 3C, 4 where X is aphosphodiester linkage and MO is compound 38649 38371 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s) C_(s) A C_(s) A C_(s)T C_(s) C_(s) Structure III of Figure 3C, 4 where X is aphosphorothioate linkage and MO is compound 38649 38458 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s) C_(s) A C_(s) A C_(s)T C_(s) C_(s) Structure I of Figure 3C, where 4 X₂ is a phophorothioatelinkage, m is 1, N_(m) is a β-D- deoxynucleoside (dA), X₁ is aphosphorothioate linkage, and MO is compound 38649 38459 A_(E)^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s) C_(s) AC_(s) A C_(s) T C_(s) C_(s) Structure I of Figure 3C, where 4 X₂ is aphophodiester linkage, m is 1, N_(m), is a β-D- deoxynucleoside (dA), X₁is a phosphorothioate linkage, and MO is compound 38649 38597 A_(E)^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s) C_(s) AC_(s) A C_(s) T C_(s) C_(s) Structure I of Figure 3C, where 4 X₂ is aphophorothioate linkage, m is 1, N_(m) is a 2′-O- methoxyethylnucleoside, X₁ is a phosphorothioate linkage, and MO is compound 3864938598 A_(E) ^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s)C_(s) A C_(s) A C_(s) T C_(s) C_(s) Structure I of Figure 3C, where 4 X₂is a phophorothioate linkage, m is 1, N_(m) is a X₁ is aphosphorothioate linkage, and MO is compound 38649

In certain embodiments, a compound provided herein comprises a modifiednucleotide and a conjugate moiety, wherein the modified oligonucleotidehas the structure C_(S)A_(S)C_(S)A_(S)C_(S)U_(S)C_(S)C_(S) (SEQ ID NO:9), wherein the subscript “S” indicates an S-cEt and nucleosides notfollowed by a subscript are β-D-deoxyribonucleosides, and eachinternucleoside linkage is a phosphorothioate internucleoside linkage,and wherein the conjugate moiety is linked to the 3′ terminus of themodified oligonucleotide and has the structure:

wherein X₁ and X₂ are phosphodiester linkages; m is 1; N is aβ-D-deoxyriboadenosine; and MO is the modified oligonucleotide.

Additional moieties for conjugation to a modified oligonucleotideinclude phenazine, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. In certain embodiments, aconjugate group is attached directly to a modified oligonucleotide.

Certain Metabolic Products

Upon exposure to exonucleases and/or endonucleases in vitro or in vivo,compounds may undergo cleavage at various positions throughout thecompound. The products of such cleavage may retain some degree of theactivity of the parent compound, and as such are considered activemetabolites. As such, a metabolic product of a compound may be used inthe methods described herein. In certain embodiments, a modifiedoligonucleotide (unconjugated or conjugated) undergoes cleavage at the5′ end and/or the 3′ end, resulting in a metabolic product that has 1,2, or 3 fewer nucleotides at the 5′ end and/or the 3′ end, relative tothe parent modified oligonucleotide. In certain embodiments, a modifiedoligonucleotide undergoes cleavage at the 5′ end, releasing the5′-terminal nucleotide and resulting in a metabolic product that has 1less nucleotide at the 5′ end, relative to the parent modifiedoligonucleotide. In certain embodiments, a modified oligonucleotideundergoes cleavage at the 5′ end, releasing two 5′-terminal nucleosidesand resulting in a metabolic product that has two fewer nucleotides atthe 5′ end, relative to the parent modified oligonucleotide. In certainembodiments, a modified oligonucleotide undergoes cleavage at the 3′end, releasing the 3′-terminal nucleotide and resulting in a metabolicproduct that has one less nucleotide at the 3′ end, relative to theparent modified oligonucleotide. In certain embodiments, a modifiedoligonucleotide undergoes cleavage at the 3′ end, releasing two3′-terminal nucleosides and resulting in a metabolic product that hastwo fewer nucleotides at the 3′ end, relative to the parent modifiedoligonucleotide.

Compounds comprising modified oligonucleotide linked to a conjugatemoiety may also undergo cleavage at a site within the linker between themodified oligonucleotide and the ligand. In certain embodiments,cleavage yields the parent modified oligonucleotide comprising a portionof the conjugate moiety. In certain embodiments, cleavage yields theparent modified oligonucleotide comprising one or more subunits of thelinker between the modified oligonucleotide and the ligand. For example,where a compound has the structure L_(n)-linker-N_(m)-P-MO, in someembodiments, cleavage yields the parent modified oligonucleotidecomprising one or more nucleotides of N_(m). In some embodiments,cleavage of a conjugated modified oligonucleotide yields the parentmodified oligonucleotide. In some such embodiments, for example, where acompound has the structure L_(n)-linker-N_(m)-P-MO, in some embodiments,cleavage yields the parent modified oligonucleotide without any of thenucleotides of N_(m).

Certain Nucleobase Sequences

Nucleobase sequences of mature miR-122 and its corresponding stem-loopsequence are found in miRBase, an online searchable database of microRNAsequences and annotation, found at microrna.sanger.ac.uk. Entries in themiRBase Sequence database represent a predicted hairpin portion of amicroRNA transcript (the stem-loop), with information on the locationand sequence of the mature microRNA sequence. The microRNA stem-loopsequences in the database are not strictly precursor microRNAs(pre-microRNAs), and may in some instances include the pre-microRNA andsome flanking sequence from the presumed primary transcript. ThemicroRNA nucleobase sequences described herein encompass any version ofthe microRNA, including the sequences described in Release 15.0 of themiRBase sequence database and sequences described in any earlier Releaseof the miRBase sequence database. A sequence database release may resultin the re-naming of certain microRNAs. The present invention encompassesmodified oligonucleotides that are complementary to any nucleobasesequence version of the microRNAs described herein.

In certain embodiments, each nucleobase of a modified oligonucleotidetargeted to miR-122 is capable of undergoing base-pairing with anucleobase at a corresponding position in the nucleobase sequence ofmiR-122, or a precursor thereof. In certain embodiments the nucleobasesequence of a modified oligonucleotide may have one or more mismatchedbasepairs with respect to its target microRNA or precursor sequence, andremains capable of hybridizing to its target sequence.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to the nucleobase sequence of miR-122precursor, such as miR-122 stem-loop sequence. As miR-122 is containedwithin a miR-122 precursor sequence, a modified oligonucleotide having anucleobase sequence complementary to miR-122 is also complementary to aregion of a miR-122 precursor.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to nucleobases 1 to 16, 1 to 17, 1 to 18,1 to 19, 1 to 20, 1 to 21, or 1 to 22 of SEQ ID NO: 1.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to nucleobases 2 to 16, 2 to 17, 2 to 18,2 to 19, 2 to 20, 2 to 21, or 2 to 22 of SEQ ID NO: 1.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence that is complementary to nucleobases 3 to 17, 3 to 18, 3 to 19,3 to 20, 3 to 21, or 3 to 22 of SEQ ID NO: 1.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is less than the length of the miR-122, or a precursorthereof. In certain such embodiments, the oligonucleotide has anucleobase sequence that is complementary to a region of miR-122, or aprecursor thereof. A modified oligonucleotide having a number of linkednucleosides that is less than the length of miR-122, wherein eachnucleobase of a modified oligonucleotide is complementary to eachnucleobase at a corresponding position in a miR-122 nucleobase sequence,is considered to be a modified oligonucleotide having a nucleobasesequence that is fully complementary to miR-122. For example, a modifiedoligonucleotide consisting of 19 linked nucleosides, where thenucleobases of nucleosides 1 through 19 are each complementary to acorresponding position of miR-122, where the miR-122 is 22 nucleobasesin length, is fully complementary to 19 contiguous nucleobases ofmiR-122. Such a modified oligonucleotide has a nucleobase sequence thatis 100% complementary to the nucleobase sequence of miR-122.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is one less than the length of the miR-122. In certainembodiments, a modified oligonucleotide has one less nucleoside at the5′ terminus. In certain embodiments, a modified oligonucleotide has oneless nucleoside at the 3′ terminus. In certain embodiments, a modifiedoligonucleotide has two fewer nucleosides at the 5′ terminus. In certainembodiments, a modified oligonucleotide has two fewer nucleosides at the3′ terminus

In certain embodiments, 15 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 15 contiguous nucleobases ofmiR-122. In certain embodiments, 16 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 16 contiguous nucleobases ofmiR-122. In certain embodiments, 17 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 17 contiguous nucleobases ofmiR-122. In certain embodiments, 18 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 18 contiguous nucleobases ofmiR-122. In certain embodiments, 19 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 19 contiguous nucleobases ofmiR-122. In certain embodiments, 20 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 20 contiguous nucleobases ofmiR-122. In certain embodiments, 21 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 21 contiguous nucleobases ofmiR-122. In certain embodiments, 22 contiguous nucleobases of a modifiedoligonucleotide are each complementary to 22 contiguous nucleobases ofmiR-122.

In certain embodiments, a modified oligonucleotide comprises anucleobase sequence that is complementary to a seed sequence, i.e. amodified oligonucleotide comprises a seed-match sequence. In certainembodiments, a seed sequence is a hexamer seed sequence. In certain suchembodiments, a seed sequence is nucleobases 1-6 of miR-122. In certainsuch embodiments, a seed sequence is nucleobases 2-7 of miR-122. Incertain such embodiments, a seed sequence is nucleobases 3-8 of miR-122.In certain embodiments, a seed sequence is a heptamer seed sequence. Incertain such embodiments, a heptamer seed sequence is nucleobases 1-7 ofmiR-122. In certain such embodiments, a heptamer seed sequence isnucleobases 2-8 of miR-122. In certain embodiments, the seed sequence isan octamer seed sequence. In certain such embodiments, an octamer seedsequence is nucleobases 1-8 of miR-122. In certain embodiments, anoctamer seed sequence is nucleobases 2-9 of miR-122.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is greater than the length the miR-122 sequence. Incertain such embodiments, the nucleobase of an additional nucleoside iscomplementary to a nucleobase of miR-122 stem-loop sequence. In certainembodiments, the number of linked nucleosides of a modifiedoligonucleotide is one greater than the length of miR-122. In certainsuch embodiments, the additional nucleoside is at the 5′ terminus of amodified oligonucleotide. In certain such embodiments, the additionalnucleoside is at the 3′ terminus of a modified oligonucleotide. Incertain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is two greater than the length of miR-122. In certainsuch embodiments, the two additional nucleosides are at the 5′ terminusof a modified oligonucleotide. In certain such embodiments, the twoadditional nucleosides are at the 3′ terminus of a modifiedoligonucleotide. In certain such embodiments, one additional nucleosideis located at the 5′ terminus and one additional nucleoside is locatedat the 3′ terminus of a modified oligonucleotide. In certainembodiments, a region of the oligonucleotide may be fully complementaryto the nucleobase sequence of miR-122, but the entire modifiedoligonucleotide is not fully complementary to miR-122. For example, amodified oligonucleotide consisting of 23 linked nucleosides, where thenucleobases of nucleosides 1 through 22 are each complementary to acorresponding position of miR-122 that is 22 nucleobases in length, hasa 22 nucleoside portion that is fully complementary to the nucleobasesequence of miR-122.

In certain embodiments, a compound comprises a modified oligonucleotideattached to a ligand through a linker comprising one or morenucleosides. For the purposes of calculating percentage complementarity,any additional nucleosides of the linker are considered to be part ofthe linker and not part of the modified oligonucleotide. Accordingly,the nucleobase sequence of the modified oligonucleotide of a conjugatedcompound may still be 100% complementary to miR-122, even where thelinker comprises one or more nucleosides that are not complementary tomiR-122.

The miR-122 nucleobase sequences set forth herein, including but notlimited to those found in the examples and in the sequence listing, areindependent of any modification to the nucleic acid. As such, nucleicacids defined by a SEQ ID NO may comprise, independently, one or moremodifications to one or more sugar moieties, to one or moreinternucleoside linkages, and/or to one or more nucleobases.

Although the sequence listing accompanying this filing identifies eachnucleobase sequence as either “RNA” or “DNA” as required, in practice,those sequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides issomewhat arbitrary. For example, a modified oligonucleotide comprising anucleoside comprising a 2′-OH sugar moiety and a thymine base could bedescribed as a DNA having a modified sugar (2′-OH for the natural 2′-Hof DNA) or as an RNA having a modified base (thymine (methylated uracil)for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to, those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,a modified oligonucleotide having the nucleobase sequence “ATCGATCG”encompasses any oligonucleotide having such nucleobase sequence, whethermodified or unmodified, including, but not limited to, such compoundscomprising RNA bases, such as those having sequence “AUCGAUCG” and thosehaving some DNA bases and some RNA bases such as “AUCGATCG” andoligonucleotides having other modified bases, such as “AT′CGAUCG,”wherein ^(Me)C indicates a 5-methylcytosine. Similarly, a modifiedoligonucleotide having the nucleobase sequence “AUCGAUCG” encompassesany oligonucleotide having such nucleobase sequence, whether modified orunmodified, including, but not limited to, such compounds comprising DNAbases, such as those having sequence “ATCGATCG” and those having someDNA bases and some RNA bases such as “AUCGATCG” and oligonucleotideshaving other modified bases, such as “AT′CGAUCG,” wherein ^(me)Cindicates a 5-methylcytosine.

Certain Uses of miR-122 Compositions

The microRNA miR-122 is a liver-expressed microRNA that is a criticalendogenous “host factor” for the replication of HCV, andoligonucleotides targeting miR-122 block HCV replication (Jopling et al.(2005) Science 309, 1577-81) Inhibition of miR-122 in chimpanzeeschronically infected with the Hepatitis C virus reduced HCV RNA level.In HCV-infected patients, inhibition of miR-122 resulted in a mean 2 logreduction in HCV RNA level after 5 weekly doses of anti-miR-122compound. The compounds described herein are potent inhibitors ofmiR-122 activity. Accordingly, provided here are methods for thetreatment of HCV infection, comprising a compound provided herein to anHCV-infected subject.

Provided herein are methods for treating an HCV-infected subjectcomprising administering to the subject a compound provided herein. Incertain embodiments, the methods provided herein comprise selecting anHCV-infected subject. In certain embodiments, the subject is a human.

In certain embodiments, the administering reduces the symptoms of HCVinfection. Symptoms of HCV infection include, without limitation, painover the liver, jaundice, nausea, loss of appetite, and fatigue.

Following an HCV treatment regimen, an HCV-infected subject mayexperience a decrease in HCV RNA level, followed by an increase in HCVRNA level, which subsequent increase is known as a rebound in HCV RNAlevel. In certain embodiments, the compounds and methods provided hereinprevent a rebound in HCV RNA level. In certain embodiments, thecompounds and methods provided herein delay a rebound in HCV RNA level.

HCV RNA level may be used to diagnose HCV infection, monitor diseaseactivity and monitor a subject's response to treatment. In certainembodiments, administering a compound provided herein reduces HCV RNAlevel. In certain embodiments, a compound herein is administered at adose that is sufficient to reduce HCV RNA level. In certain embodiments,the methods provided herein comprise selecting a subject having an HCVRNA level greater than 350,000 copies per milliliter of serum, between350,000 and 3,500,000 copies per milliliter of serum, or greater than3,500,000 copies per milliliter of serum. In certain embodiments, themethods provided herein comprise reducing HCV RNA level. In certainembodiments, the methods provided herein comprise reducing HCV RNA levelto below 200 copies per milliliter of serum, to below 100 copies permilliliter of serum, or to below 40 copies per milliliter of serum. HCVRNA level may be referred to as “viral load” or “HCV RNA titer.”

Changes to HCV RNA level may be described as log changes. For example, adrop from 60,000 to 600 would be a 2-log drop in HCV RNA level. Incertain embodiments, the methods provided herein achieve a HCV RNA leveldecrease greater than or equal to 2 logs. In certain embodiments, themethods provided herein achieve an HCV RNA level decrease of at least0.5 fold, at least 1.0 fold, at least 1.5 fold, at least 2.0 fold, or atleast 2.5 fold.

In certain embodiments, the methods provided herein comprise achieving asustained virological response.

HCV-infected subjects may develop HCV-associated diseases. The majorhepatological consequence of HCV infection is cirrhosis andcomplications thereof including hemorrhage, hepatic insufficiency, andhepatocellular carcinoma. An additional complication is fibrosis, whichis the result of chronic inflammation causing the deposition ofextracellular matrix component, which leads to distortion of the hepaticarchitecture and blockage of the microcirculation and liver function. Ascirrhosis progresses and the fibrotic tissue builds up, severenecroinflammatory activity ensues and steatosis begins. Steatosis leadsto extrahepatic pathologies including diabetes, protein malnutrition,hypertension, cell toxins, obesity, and anoxia. As fibrosis andsteatosis becomes severe the liver will eventually fail and requireliver transplantation. HCV-infected subjects may also develophepatocellular carcinoma. In certain embodiments, an HCV-infectedsubject has an HCV-associated disease. In certain embodiments, theHCV-associated disease is cirrhosis, fibrosis, steatohepatitis,steatosis, and/or hepatocellular carcinoma.

In certain embodiments, an HCV-infected subject has one or morediseases. In certain embodiments, an HCV-infected subject is infectedwith one or more viruses other than HCV. In certain embodiments, anHCV-infected subject is infected with human immunodeficiency virus(HIV). The compounds provided herein may be concomitantly administeredwith one or more additional therapeutic agents. In certain embodiments,the one or more additional therapeutic agents comprises an immunetherapy, an immunomodulator, therapeutic vaccine, antifibrotic agent,anti-inflammatory agent, bronchodilator, mucolytic agent,anti-muscarinic, anti-leukotriene, inhibitor of cell adhesion,anti-oxidant, cytokine agonist, cytokine antagonist, lung surfactant,antimicrobial, anti-viral agent, anti-HCV agent, an anti-cancer agent,an anti-miR-122 compound, an RNAi agent or a cyclophilin inhibitor.

In certain embodiments, the one or more additional therapeutic agentsmay be selected from a protease inhibitor, a polymerase inhibitor, acofactor inhibitor, an RNA polymerase inhibitor, a structural proteininhibitor, a non-structural protein inhibitor, a cyclophilin inhibitor,an entry inhibitor, a TLR7 agonist, and an interferon.

In certain embodiments, the additional therapeutic agent is a modifiedoligonucleotide having the structureC_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) (SEQ ID NO: 7), wherenucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “L”indicate LNA nucleosides; and each internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, atherapeutic agent is a GalNAc-conjugatedC_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) (SEQ ID NO: 7). In someembodiments, all of the C_(L) nucleosides are ^(Me)C_(L) nucleosides,wherein the superscript “Me” indicates 5-methylcytosine.

In certain embodiments, the additional therapeutic agent is selectedfrom a protease inhibitor, an NS5A inhibitor, an NS3/4A inhibitor, anucleoside NS5B inhibitor, a nucleotide NS5B inhibitor, a non-nucleosideNS5B inhibitor, a cyclophilin inhibitor and an interferon.

In certain embodiments, the additional therapeutic agent is selectedfrom interferon alfa-2a, interferon alpha-2b, interferon alfacon-1,peginterferon alpha-2b, peginterferon alpha-2a, interferon-alpha-2bextensed release, interferon lambda, sofosbuvir, ribavirin, telapravir,boceprevir, vaniprevir, asunaprevir, ritonavir, setrobuvir, daclastavir,simeprevir, alisporivir, mericitabine, tegobuvir, danoprevir,sovaprevir, and neceprevir. In certain embodiments, the additionaltherapeutic agent is selected from faldaprevir, ABT-450, MK-5172,mericitabine, ledipasvir, ombitasvir, GS-5816, MK-8742, dasabuvir,BMS-791325, and ABT-072.

In certain embodiments, the additional therapeutic agent is selectedfrom an interferon, ribavirin, and telapravir. In certain embodiments,the interferon is selected from interferon alfa-2a, interferon alpha-2b,interferon alfacon-1, peginterferon alpha-2b, and peginterferonalpha-2a.

In certain embodiments, the additional therapeutic agent includespeginterferon alpha-2b and ribavirin. For example, a subject may receivea therapy that comprises a compound provided herein, peginterferonalpha-2b and ribavirin. In certain embodiments, the at least oneadditional therapeutic agent includes peginterferon alpha-2a andribavirin. For example, a subject may receive a therapy that comprises acompound provided herein, peginterferon alpha-2a and ribavirin. Incertain embodiments, the additional therapeutic agents are ombitasvirand ABT-450. In certain embodiments, the additional therapeutic agentsare asunaprevir, daclatasvir, and BMS-791325. In certain embodiments,the additional therapeutic agents are sofosbuvir and ledipasivr. Incertain embodiments, the additional therapeutic agents are MK-8742 andMK-5172.

Certain subjects receiving a certain therapy, for example interferon orribaviran therapy, may not experience a significant or therapeuticallybeneficial reduction in HCV RNA level. Such subjects may benefit fromadministration of one or more additional therapeutic agents. In certainembodiments, a subject of the methods provided herein is anon-responder. In certain embodiments, a subject is an interferonnon-responder. In certain embodiments, a subject is a direct-actinganti-viral non-responder.

In certain embodiments, an additional therapeutic agent is an anti-viralagent used in the treatment of HIV infection. In certain embodiments, anadditional therapeutic agent is a non-nucleoside reverse transcriptaseinhibitors (NNRTIs). In certain embodiments, an additional therapeuticagent is a nucleoside reverse transcriptase inhibitors (NRTIs). Incertain embodiments, an additional therapeutic agent is a proteaseinhibitor. In certain embodiments, an additional therapeutic agent is anentry inhibitor or fusion inhibitor. In certain embodiments, anadditional therapeutic agent is an integrase inhibitor. In certainembodiments, an additional therapeutic agent is selected from efavirenz,etravirine, nevirapine, abacavir, emtricitabine, tenofovir, lamivudine,zidovudine, atazanavir, darunavir, fosamprenavir, ritonavir,enfuvirtide, maraviroc, and raltegravir.

A subject infected with HCV may experience abnormal liver function,which is assessed by measuring one or more of bilirubin, albumin, andprothombin time. Measurement of the liver enzymes alanineaminotransferase (ALT), and aspartate aminotransferase (AST) isperformed to assess liver inflammation. One or more abnormal levels ofthese markers may indicate abnormal liver function. In certainembodiments, the methods provided herein comprise normalizing liverfunction. In certain embodiments, the methods provided herein comprisenormalizing liver enzyme levels.

In any of the methods provided, herein, the compound may be present in apharmaceutical composition.

The compounds provided herein may be for use in therapy. In certainembodiments, the compound is for use in treating an HCV-infectedsubject. In certain embodiments, the subject is a human. The compoundfor use in treating an HCV-infected subject may, in certain embodiments,be for use in any method of treatment described herein.

Provided herein are methods comprising administering a compound providedherein to a subject having a miR-122-associated condition. In certainembodiments, a miR-122-associated condition is HCV infection.

In certain embodiments, a miR-122-associated condition is elevatedcholesterol. In certain embodiments, administration of an anti-miR-122compound to a subject results in reduced serum cholesterol. Accordingly,in certain embodiments, provided herein are methods of loweringcholesterol in a subject, comprising administering to a subject acompound provided herein. In certain embodiments, cholesterol levels maybe used as a biomarker to assess the activity of an anti-miR-122compound provided herein, alone or in addition to another indicator ofefficacy, e.g. reduction in HCV RNA levels. Accordingly, provided hereinare methods comprising administering a compound provided herein to asubject, collecting a blood sample from the subject, and measuringcholesterol in the blood sample from the subject. The level ofcholesterol may be used as an indicator of anti-miR-122 compoundactivity in the subject.

In certain embodiments, a miR-122-associated condition is steatosis.Accordingly, in certain embodiments, provided herein are methods ofreducing steatosis in a subject, comprising administering to the subjecta compound provided herein.

In certain embodiments, a miR-122-associated condition is an ironoverload disorder. An iron overload disorder may occur as a result of agenetic mutation that causes the body to absorb excess amounts of iron.An iron overload disorder may also have non-genetic causes, includingbut not limited to chronic blood transfusions, chronic hepatitis, oringestion of an excess amount of iron. In certain embodiments, an ironoverload disorder is selected from transfusional iron overload, dietaryiron overload, hereditary hemochromatosis, sickle cell disease,thalassemia, X-linked sideroblastic anemia, pyruvate kinase deficiency,and glucose-6-phosphate dehydrogenase deficiency. In certainembodiments, an iron overload disorder is a hereditary hemochromatosisselected from hemochromatosis type 1, hemochromatosis type 2A,hemochromatosis type 2B, hemochromatosis type 3, hemochromatosis type 4(or ferroportin disease), African hemochromatosis, neonatalhemochromatosis, aceruloplasminemia, and atransferrinemia. In certainembodiments, administration of a compound provided herein to a subjecthaving an iron overload disorder results in reduction of excess iron inthe body of the subject.

Certain Modifications

A modified oligonucleotide may comprise one or more modifications to anucleobase, sugar, and/or internucleoside linkage. A modifiednucleobase, sugar, and/or internucleoside linkage may be selected overan unmodified form because of desirable properties such as, for example,enhanced cellular uptake, enhanced affinity for other oligonucleotidesor nucleic acid targets and increased stability in the presence ofnucleases.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleosides. In certain embodiments, a modified nucleoside is astabilizing nucleoside. An example of a stabilizing nucleoside is a2′-modified nucleoside.

In certain embodiments, a modified nucleoside comprises a modified sugarmoiety. In certain embodiments, a modified nucleoside comprising amodified sugar moiety comprises an unmodified nucleobase. In certainembodiments, a modified sugar comprises a modified nucleobase. Incertain embodiments, a modified nucleoside is a 2′-modified nucleoside.

In certain embodiments, a 2′-modified nucleoside comprises a bicyclicsugar moiety. In certain such embodiments, the bicyclic sugar moiety isa D sugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridgegroup between the 2′ and the 4′-carbon atoms. In certain suchembodiments, the bridge group comprises from 1 to 8 linked biradicalgroups. In certain embodiments, the bicyclic sugar moiety comprises from1 to 4 linked biradical groups. In certain embodiments, the bicyclicsugar moiety comprises 2 or 3 linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises 2 linked biradicalgroups. Examples of such 4′ to 2′ sugar substituents, include, but arenot limited to: —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)₃-2; 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, andanalogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)-O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)-O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprise 1or from 2 to 4 linked groups independently selected from—[C(R_(a))(R_(b))_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—,—C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. In certain embodiments, bicyclicnucleosides include, but are not limited to, (A) a-L-Methyleneoxy(4′-CH₂—O-2′) BNA; (B) β-D-Methyleneoxy (4′-CH₂—O-2′) BNA; (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA; (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA;(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA; (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt);(G) methylene-thio (4′-CH₂—S-2′) BNA; (H) methylene-amino(4′-CH₂-N(R)-2′) BNA; (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA;(J) c-MOE (4′-CH₂—OMe-2′) BNA and (K) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from halo, allyl, amino, azido, SH, CN,OCN, CF₃, OCF₃, O-, S-, or N(R_(m))-alkyl; O-, S-, or N(R_(m))-alkenyl;O-, S- or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n))or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be furthersubstituted with one or more substituent groups independently selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂),thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a 2′-modified nucleoside is a 4′-thio modifiednucleoside. In certain embodiments, a 2′-modified nucleoside is a4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-0 replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

In certain embodiments, a modified oligonucleotide comprises one or moreinternucleoside modifications. In certain such embodiments, eachinternucleoside linkage of a modified oligonucleotide is a modifiedinternucleoside linkage. In certain embodiments, a modifiedinternucleoside linkage comprises a phosphorus atom.

In certain embodiments, a modified oligonucleotide comprises at leastone phosphorothioate internucleoside linkage. In certain embodiments,each internucleoside linkage of a modified oligonucleotide is aphosphorothioate internucleoside linkage.

In certain embodiments, a modified internucleoside linkage does notcomprise a phosphorus atom. In certain such embodiments, aninternucleoside linkage is formed by a short chain alkyl internucleosidelinkage. In certain such embodiments, an internucleoside linkage isformed by a cycloalkyl internucleoside linkages. In certain suchembodiments, an internucleoside linkage is formed by a mixed heteroatomand alkyl internucleoside linkage. In certain such embodiments, aninternucleoside linkage is formed by a mixed heteroatom and cycloalkylinternucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more short chainheteroatomic internucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by one or more heterocyclicinternucleoside linkages. In certain such embodiments, aninternucleoside linkage has an amide backbone. In certain suchembodiments, an internucleoside linkage has mixed N, O, S and CH₂component parts.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleobases. In certain embodiments, a modified nucleobase isselected from 7-deazaguanine, 7-deazaadenine, hypoxanthine, xanthine,7-methylguanine, 2-aminopyridine and 2-pyridone. In certain embodiments,a modified nucleobase is selected from 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclicheterocycle. In certain embodiments, a modified nucleobase comprises atricyclic heterocycle. In certain embodiments, a modified nucleobasecomprises a phenoxazine derivative. In certain embodiments, thephenoxazine can be further modified to form a nucleobase known in theart as a G-clamp.

In certain such embodiments, the compound comprises a modifiedoligonucleotide having one or more stabilizing groups that are attachedto one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini. Cap structures include, for example,inverted deoxy abasic caps.

Suitable cap structures include a 4′,5′-methylene nucleotide, a1-(beta-D-erythrofuranosyl) nucleotide, a 4′-thio nucleotide, acarbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, anL-nucleotide, an alpha-nucleotide, a modified base nucleotide, aphosphorodithioate linkage, a threo-pentofuranosyl nucleotide, anacyclic 3′,4′-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide,an acyclic 3,5-dihydroxypentyl nucleotide, a 3′-3′-inverted nucleotidemoiety, a 3′-3′-inverted abasic moiety, a 3′-2′-inverted nucleotidemoiety, a 3′-2′-inverted abasic moiety, a 1,4-butanediol phosphate, a3′-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a3′-phosphate, a 3′-phosphorothioate, a phosphorodithioate, a bridgingmethylphosphonate moiety, and a non-bridging methylphosphonate moiety5′-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecylphosphate, a hydroxypropyl phosphate, a 5′-5′-inverted nucleotidemoiety, a 5′-5′-inverted abasic moiety, a 5′-phosphoramidate, a5′-phosphorothioate, a 5′-amino, a bridging and/or non-bridging5′-phosphoramidate, a phosphorothioate, and a 5′-mercapto moiety.

Certain Synthesis Methods

Modified oligonucleotides may be made with automated, solid phasesynthesis methods known in the art. During solid phase synthesis,phosphoramidite monomers are sequentially coupled to a nucleoside thatis covalently linked to a solid support. This nucleoside is the 3′terminal nucleoside of the modified oligonucleotide. Typically, thecoupling cycle comprises four steps: detritylation (removal of a5′-hydroxyl protecting group with acid), coupling (attachment of anactivated phosphoroamidite to the support bound nucleoside oroligonucleotide), oxidation or sulfurization (conversion of a newlyformed phosphite trimester with an oxidizing or sulfurizing agent), andcapping (acetylation of unreacted 5′-hydroxyl groups). After the finalcoupling cycle, the solid support-bound oligonucleotide is subjected toa detritylation step, followed by a cleavage and deprotection step thatsimultaneously releases the oligonucleotide from the solid support andremoves the protecting groups from the bases. The solid support isremoved by filtration, the filtrate is concentrated and the resultingsolution is tested for identity and purity. The oligonucleotide is thenpurified, for example using a column packed with anion-exchange resin.

GalNAc-conjugated modified oligonucleotides may be made with automatedsolid phase synthesis, similar to the solid phase synthesis thatproduced unconjugated oligonucleotides. During the synthesis ofGalNAc-conjugated oligonucleotides, the phosphoramidite monomers aresequentially coupled to a GalNAc conjugate which is covalently linked toa solid support. The synthesis of GalNAc conjugates and GalNAc conjugatesolid support is described, for example, in U.S. Pat. No. 8,106,022, andInternational Application Publication No. WO 2013/033230, each of whichis herein incorporated by reference in its entiretly for the descriptionof the synthesis of carbohydrate-containing conjugates, includingconjugates comprising one or more GalNAc moieties, and of the synthesisof conjugate covalently linked to solid support.

Certain Pharmaceutical Compositions

Any of the compounds provided herein may be prepared as a pharmaceuticalcomposition. In certain embodiments, a pharmaceutical composition isadministered in the form of a dosage unit (e.g., tablet, capsule, bolus,etc.). In some embodiments, a pharmaceutical composition comprises acompound provided herein at a dose within a range selected from 25 mg to800 mg, 25 mg to 700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400mg, 25 mg to 300 mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg,200 mg to 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg,600 mg to 800 mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg,250 mg to 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600mg. In certain embodiments, such pharmaceutical compositions comprise acompound provided herein present at a dose selected from 25 mg, 30 mg,35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg, 580mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg, 625mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, and 800 mg.In certain such embodiments, a pharmaceutical composition of thecomprises a dose compound provided herein selected from 25 mg, 50 mg, 75mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600mg, 700 mg, and 800 mg.

In certain embodiments, a pharmaceutical composition comprising acompound provided herein is administered at a dose of 10 mg/kg or less,9 mg/kg or less, 8 mg/kg or less, 7.5 mg/kg or less, 7 mg/kg or less,6.5 mg/kg or less, 6 mg/kg or less, 5.5 mg/kg or less, 5 mg/kg or less,4.5 mg/kg or less, 4 mg/kg or less, 3.5 mg/kg or less, 3 mg/kg or less,2.5 mg/kg or less, 2 mg/kg or less, 1.5 mg/kg or less, 1 mg/kg or less,0.75 mg/kg or less, 0.5 mg/kg or less, or 0.25 mg/kg or less.

In certain embodiments, a pharmaceutical agent is sterile lyophilizedcompound that is reconstituted with a suitable diluent, e.g., sterilewater for injection or sterile saline for injection. The reconstitutedproduct is administered as a subcutaneous injection or as an intravenousinfusion after dilution into saline. The lyophilized drug productconsists of a compound which has been prepared in water for injection,or in saline for injection, adjusted to pH 7.0-9.0 with acid or baseduring preparation, and then lyophilized. The lyophilized compound maybe 25-800 mg of an oligonucleotide. It is understood that thisencompasses 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 725, 750, 775, and 800 mg of modified lyophilized oligonucleotide.Further, in some embodiments, the lyophilized compound is present in anamount that ranges from 25 mg to 800 mg, 25 mg to 700 mg, 25 mg to 600mg, 25 mg to 500 mg, 25 mg to 400 mg, 25 mg to 300 mg, 25 mg to 200 mg,25 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800 mg, 300 mg to 800 mg,400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800 mg, 100 mg to 700 mg,150 mg to 650 mg, 200 mg to 600 mg, 250 mg to 550 mg, 300 mg to 500 mg,300 mg to 400 mg, or 400 mg to 600 mg. The lyophilized drug product maybe packaged in a 2 mL Type I, clear glass vial (ammoniumsulfate-treated), stoppered with a bromobutyl rubber closure and sealedwith an aluminum FLIP-OFF® overseal.

In certain embodiments, a pharmaceutical composition provided hereincomprises a compound in a therapeutically effective amount. In certainembodiments, the therapeutically effective amount is sufficient toprevent, alleviate or ameliorate symptoms of a disease or to prolong thesurvival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, the pharmaceutical compositions provided hereinmay additionally contain other adjunct components conventionally foundin pharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In one method, the nucleic acid is introduced into preformedliposomes or lipoplexes made of mixtures of cationic lipids and neutrallipids. In another method, DNA complexes with mono- or poly-cationiclipids are formed without the presence of a neutral lipid. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to a particular cell or tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to fat tissue. In certain embodiments, a lipidmoiety is selected to increase distribution of a pharmaceutical agent tomuscle tissue.

In certain embodiments, INTRALIPID is used to prepare a pharmaceuticalcomposition comprising an oligonucleotide. Intralipid is fat emulsionprepared for intravenous administration. It is made up of 10% soybeanoil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water forinjection. In addition, sodium hydroxide has been added to adjust the pHso that the final product pH range is 6 to 8.9.

In certain embodiments, a pharmaceutical composition provided hereincomprises a polyamine compound or a lipid moiety complexed with anucleic acid. Such preparations are described in PCT publicationWO/2008/042973, which is herein incorporated by reference in itsentirety for the disclosure of lipid preparations. Certain additionalpreparations are described in Akinc et al., Nature Biotechnology 26,561-569 (1 May 2008), which is herein incorporated by reference in itsentirety for the disclosure of lipid preparations.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more compounds and one or more excipients. In certainsuch embodiments, excipients are selected from water, salt solutions,alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesiumstearate, talc, silicic acid, viscous paraffin, hydroxymethylcelluloseand polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein isprepared using known techniques, including, but not limited to mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or tableting processes.

In certain embodiments, a pharmaceutical composition provided herein isa liquid (e.g., a suspension, elixir and/or solution). In certain ofsuch embodiments, a liquid pharmaceutical composition is prepared usingingredients known in the art, including, but not limited to, water,glycols, oils, alcohols, flavoring agents, preservatives, and coloringagents.

In certain embodiments, a pharmaceutical composition provided herein isa solid (e.g., a powder, tablet, and/or capsule). In certain of suchembodiments, a solid pharmaceutical composition comprising one or moreoligonucleotides is prepared using ingredients known in the art,including, but not limited to, starches, sugars, diluents, granulatingagents, lubricants, binders, and disintegrating agents.

In certain embodiments, a pharmaceutical composition provided herein isformulated as a depot preparation. Certain such depot preparations aretypically longer acting than non-depot preparations. In certainembodiments, such preparations are administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more compounds provided herein to specific tissues orcell types. For example, in certain embodiments, pharmaceuticalcompositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided hereincomprises a sustained-release system. A non-limiting example of such asustained-release system is a semi-permeable matrix of solid hydrophobicpolymers. In certain embodiments, sustained-release systems may,depending on their chemical nature, release pharmaceutical agents over aperiod of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain of such embodiments, apharmaceutical composition is formulated by combining one or morecompounds comprising a modified oligonucleotide with one or morepharmaceutically acceptable carriers. Certain of such carriers enablepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject. In certain embodiments, pharmaceuticalcompositions for oral use are obtained by mixing oligonucleotide and oneor more solid excipient. Suitable excipients include, but are notlimited to, fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certainembodiments, such a mixture is optionally ground and auxiliaries areoptionally added. In certain embodiments, pharmaceutical compositionsare formed to obtain tablets or dragee cores. In certain embodiments,disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate) are added.

In certain embodiments, dragee cores are provided with coatings. Incertain such embodiments, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to tablets or dragee coatings.

In certain embodiments, pharmaceutical compositions for oraladministration are push-fit capsules made of gelatin. Certain of suchpush-fit capsules comprise one or more pharmaceutical agents of thepresent invention in admixture with one or more filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In certain embodiments,pharmaceutical compositions for oral administration are soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In certain soft capsules, one or more pharmaceutical agents ofthe present invention are be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

In certain embodiments, pharmaceutical compositions are prepared forbuccal administration. Certain of such pharmaceutical compositions aretablets or lozenges formulated in conventional manner

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, one or more modified oligonucleotides providedherein is administered as a prodrug. In certain embodiments, upon invivo administration, a prodrug is chemically or enzymatically convertedto the biologically, pharmaceutically or therapeutically more activeform of an oligonucleotide. In certain embodiments, prodrugs are usefulbecause they are easier to administer than the corresponding activeform. For example, in certain instances, a prodrug may be morebioavailable (e.g., through oral administration) than is thecorresponding active form. In certain embodiments, prodrugs possesssuperior transmittal across cell membranes. In certain embodiments, aprodrug facilitates delivery of a modified oligonucleotide to thedesired cell type, tissue, or organ. In certain embodiments, a prodrugis a compound comprising a conjugated modified oligonucleotide. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certainembodiments, a prodrug is an ester. In certain such embodiments, theester is metabolically hydrolyzed to carboxylic acid uponadministration. In certain instances the carboxylic acid containingcompound is the corresponding active form. In certain embodiments, aprodrug comprises a short peptide (polyaminoacid) bound to an acidgroup. In certain of such embodiments, the peptide is cleaved uponadministration to form the corresponding active form. In certainembodiments, a prodrug is produced by modifying a pharmaceuticallyactive compound such that the active compound will be regenerated uponin vivo administration. The prodrug can be designed to alter themetabolic stability or the transport characteristics of a drug, to maskside effects or toxicity, to improve the flavor of a drug or to alterother characteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392).

Certain Routes of Administration

In certain embodiments, administering to a subject comprises parenteraladministration. In certain embodiments, administering to a subjectcomprises intravenous administration. In certain embodiments,administering to a subject comprises subcutaneous administration.

In certain embodiments, administering to a subject comprisesintraarterial, pulmonary, oral, rectal, transmucosal, intestinal,enteral, topical, transdermal, suppository, intrathecal,intraventricular, intraperitoneal, intranasal, intraocular,intramuscular, intramedullary, and intratumoral administration.

Certain miR-122 Kits

The present invention also provides kits. In some embodiments, the kitscomprise one or more compounds provided herein. In some embodiments, acompound provided herein is present within a vial. A plurality of vials,such as 10, can be present in, for example, dispensing packs. In someembodiments, the vial is manufactured so as to be accessible with asyringe. The kit can also contain instructions for using the compoundsprovided herein.

In some embodiments, the kits may be used for administration of acompound provided herein to a subject. In such instances, in addition tocomprising at least one compound provided herein, the kit can furthercomprise one or more of the following: syringe, alcohol swab, cottonball, and/or gauze pad. In some embodiments, the compounds complementaryto miR-122 can be present in a pre-filled syringe (such as a single-dosesyringes with, for example, a 27 gauge, ½ inch needle with a needleguard), rather than in a vial. A plurality of pre-filled syringes, suchas 10, can be present in, for example, dispensing packs. The kit canalso contain instructions for administering a compound provided herein.

Certain Experimental Models

In certain embodiments, the present invention provides methods of usingand/or testing a compound provided herein in an experimental model.Those having skill in the art are able to select and modify theprotocols for such experimental models to evaluate a compound providedherein.

The effects of antisense inhibition of a microRNA following theadministration of anti-miR compounds may be assessed by a variety ofmethods known in the art. In certain embodiments, these methods are beused to quantitate microRNA levels in cells or tissues in vitro or invivo. In certain embodiments, changes in microRNA levels are measured bymicroarray analysis. In certain embodiments, changes in microRNA levelsare measured by one of several commercially available PCR assays, suchas the TaqMan® MicroRNA Assay (Applied Biosystems, a Life Technologiesbrand).

In vitro activity of anti-miR compounds may be assessed using aluciferase cell culture assay. In this assay, a microRNA luciferasesensor construct is engineered to contain one or more binding sites ofthe microRNA of interest fused to a luciferase gene. When the microRNAbinds to its cognate site in the luciferase sensor construct, luciferaseexpression is suppressed. When the appropriate anti-miR is introducedinto the cells, it binds to the target microRNA and relieves suppressionof luciferase expression. Thus, in this assay anti-miRs that areeffective inhibitors of the microRNA of interest will cause an increasein luciferase expression.

Activity of anti-miR compounds may be assessed by measuring the mRNAand/or protein level of a target of a microRNA. A microRNA binds to acomplementary site within one or more target RNAs, leading tosuppression of a target RNA, thus inhibition of the microRNA results inthe increase in the level of mRNA and/or protein of a target of themicroRNA (i.e., derepression). The derepression of one or more targetRNAs may be measured in vivo or in vitro. For example, a target ofmiR-122 is aldolase A (ALDOA) Inhibition of miR-122 results in anincrease in the level of ALDOA mRNA, thus ALDOA mRNA levels may be usedto evaluate the inhibitory activity of an anti-miR-122 compound.

The effects of anti-miR-122 compounds on HCV replication may be measuredin an HCV replicon assay. In this assay, compounds are introduced into acell line (e.g., a human hepatoma cell line) that contains a subgenomicreplicon of HCV with a stable luciferase reporter and three cellculture-adaptive mutations (luc-ubi-neo/ET). The luciferase reporter isused as an indirect measure of HCV replication. The replicon used may bea parent HCV genotype or an HCV genotype with mutations that conferresistance to anti-viral agents. Anti-miR-122 compounds may be evaluatedalone or in combination with other agents used in the treatment ofHCV-infection. In some embodiments, a modified oligonucleotide may betested in an in vivo or in vitro assay, and subsequently conjugated toform a compound for use in the methods described herein.

EXAMPLES

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. Those of ordinaryskill in the art will readily adopt the underlying principles of thisdiscovery to design various compounds without departing from the spiritof the current invention.

Example 1 Design and Evaluation of Anti-miR-122 Compounds

To identify potent inhibitors of miR-122, numerous anti-miR-122 modifiedoligonucleotides were designed and synthesized. The modifiedoligonucleotides varied in length, and in the number, placement, andidentity of bicyclic nucleosides and non-bicyclic nucleosides. Thecompounds were evaluated in a number of assays, to identify anti-miRsthat are suitable therapeutic agents for the treatment of HCV infection.The evaluation of the compounds was performed in an iterative manner, inwhich highly active compounds were further optimized through designchanges, and the resultant compounds were then subjected to additionalscreening. The compound evaluation process included assessment ofpotency, safety, and physicochemical characteristics.

In total, over 400 anti-miR-122 modified oligonucleotides were designedand tested in a first luciferase cell culture activity assay. Followingan additional luciferase assay and for certain compounds measurement ofmetabolic stability, approximately 70 of these compounds were selectedfor further in vivo testing. Of these 70 compounds approximately 10compounds were identified as having a suitable in vivo potency (e.g. anED₅₀ of less than 5 mg/kg). A subset of these compounds was identifiedas having a certain safety profile in rodents and non-human primates.Thus, of the hundreds of compounds screened, only a small subset of theinitial over 400 compounds met certain potency, safety andphysicochemical criteria.

Certain anti-miR-122 compounds are shown in Table A. The “position onmiR-122” is the position to which the nucleoside in that column iscomplementary to SEQ ID NO: 1, counting from the 5′ end SEQ ID NO: 1.

TABLE A Certain Anti-miR-122 Compounds Cmpd # Position on miR- SEQUENCE(5′ to 3′) and MODIFICATIONS SEQ ID 122 22 21 20 19 18 17 16 15 14 13 1211 10 9 8 7 6 5 4 3 2 1 NO 38011 C_(s) C A U_(s) T G_(s) U_(s) C A C_(s)A C_(s) T C_(s) C_(s) A 3 38012 C_(s) C A_(s) T T G U_(s) C_(s) A C_(s)A C_(s) T C_(s) C_(s) A 3 38013 C_(s) C A U_(s) T G_(s) T C_(s) A C_(s)A C_(s) T C_(s) C_(s) A 3 38014 C_(s) C A U_(s) T G_(s) U_(s) C A C_(s)A C_(s) T C_(s) C_(s) A_(E) 3 38015 ^(Me)C_(s) C A T_(s) T G_(s) T_(s) CA ^(Me)C_(s) A ^(Me)C_(s) T ^(Me)C_(s) ^(Me)C_(s) A_(E) 3 38016^(Me)C_(s) C A T_(s) T G T_(s) ^(Me)C_(s) A ^(Me)C_(s) A ^(Me)C_(s) T^(Me)C_(s) ^(Me)C_(s) A_(E) 3 38021 C_(L) C A T_(L) T G T_(L) C_(L) AC_(L) A C_(L) T C_(L) C_(L) 4 38646 A_(E) ^(Me)C_(E) A_(E) ^(Me)C_(E) CA_(s) T T G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s) 4 38647 A_(E)^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(s) T T G U_(s) C_(s) A C_(s) AC_(s) T C_(s) C_(s) 4 38648 A_(E) ^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E)A_(E) T T G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s) 4 38649 A_(E)^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s) C_(s) AC_(s) A C_(s) T C_(s) C_(s) 4 38650 A_(E) ^(Me)C_(E) A_(E) ^(Me)C_(E)^(Me)C_(E) A_(E) T_(E) T_(E) G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s)4 38651 A_(E) ^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T_(E)G_(E) U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s) 4 38652 ^(Me)C_(E) A_(E)A_(E) A_(E) ^(Me)C_(E) A_(E) C_(s) C A_(s) T T G U_(s) C_(s) A C_(s) AC_(s) T C_(s) C_(s) 5 38660 ^(Me)C_(E) A_(E) A_(E) A_(E) ^(Me)C_(E)A_(E) C_(s) C A_(s) T T G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s)T_(E) 6 38872 C_(s) C A U_(s) T G U_(s) C_(s) A C_(s) A C_(s) T C_(s)C_(s) A 3 38910 ^(Me)C_(E) C_(s) A U_(s) T G U_(s) C_(s) A C_(s) A C_(s)T C_(s) C_(s) A_(E) 3

Sugar moieties are indicated as follows: nucleosides not followed by asubscript indicate β-D-deoxyribonucleosides; nucleosides followed by asubscript “E” indicate 2′-MOE nucleosides; nucleosides followed by asubscript “S” indicate S-cEt nucleosides; nucleosides followed by asubscript “L” indicate LNA nucleosides. Each internucleoside linkage isa phosphorothioate internucleoside linkage. Superscript “Me” indicates a5-methyl group on the base of the nucleoside.

Potency In Vitro and In Vivo Potency

An in vitro luciferase assay was used to measure the ability of eachcompound to inhibit the activity of miR-122 in cell culture. In thisassay, a microRNA luciferase sensor construct was engineered to containmultiple miR-122 binding sites fused to a luciferase gene. When miR-122binds to its targetsites in the luciferase sensor construct, luciferaseexpression is suppressed. When an active anti-miR-122 compound isintroduced into the cells, it binds to miR-122 and relieves suppressionof luciferase expression. Thus, in this assay anti-miR-122 compoundsthat are effective inhibitors of the miR-122 will cause an increase inluciferase expression.

The luciferase sensor construct, and a second construct expressingmiR-122, were introduced into Hela cells. Anti-miR-122 compounds weretransfected into the cells at several different concentrations.Compounds with an EC₅₀ of less than 100 nM were subjected to anadditional luciferase assay, at a broader range of anti-miRconcentrations than in the initial luciferase assay, to confirmactivity. Compounds were tested in two separate experiments, asindicated in Table B. The mean EC50 for each compound is shown in TableB. The results demonstrate that alterations to sugar moiety ornucleobase can impact in vitro potency of an anti-miR-122 compound.

TABLE B Mean EC50 in the luciferase cell culture assay Luciferase SEQ IDmean Compound # Sequence and Chemistry NO Experiment # EC₅₀ 38011C_(S)CAU_(S)TG_(S)U_(S)CAC_(S)AC_(S)TC_(S)C_(S)A 3 1 38.45 38012C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 1 43.78 38013C_(S)CAU_(S)TG_(S)TC_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 1 53.27 38014C_(S)CAU_(S)TG_(S)U_(S)CAC_(S)AC_(S)TC_(S)C_(S)A_(E) 3 1 42.71 38015^(Me)C_(S)CAT_(S)TG_(S)T_(S)CA^(Me)C_(S)A^(Me)C_(S)T^(Me)C_(S)^(Me)C_(S)A_(E) 3 1 42.40 38016 ^(Me)C_(S)CAT_(S)TGT_(S)^(Me)C_(S)A^(Me)C_(S)A^(Me)C_(S)T^(Me)C_(S) ^(Me)C_(S)A_(E) 3 1 14.0738021 ^(Me)C_(L)CAT_(L)TGT_(L)^(Me)C_(L)A^(Me)C_(L)A^(Me)C_(L)T^(Me)C_(L) ^(Me)C_(L)A_(E) 3 1 11.1838872 C_(S)CAU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 2 18.3 38910^(Me)CC_(S)AU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A_(E) 3 Not tested38646 A_(E) ^(Me)C_(E)A_(E)^(Me)C_(E)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 2 77.15 38647A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 2 57.44 38648A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 2 97.68 38649A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(s) 4 2 46.76 38650A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)T_(E)GU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 2 28.1638651 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)T_(E)G_(E)U_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 4 226.12 38652 ^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) 5 2 31.8638659 C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E) 10 2 130.0138660 ^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E) 6 217.02

To determine in vivo potency, certain compounds were evaluated for theirability to de-repress the expression of liver aldolase A (ALDOA), a genethat is normally suppressed by miR-122 activity. Inhibition of miR-122leads to an increase in ALDOA expression, thus ALDOA mRNA levels can beused to measure miR-122 inhibitory activity in vivo. Compounds wereadministered to mice in a single dose at the amounts indicated in TableC, and after 7 days the study was terminated, and ALDOA mRNA levels weremeasured, by quantitative PCR, in RNA isolated from liver. Except forcompound 38910, each compound in Table C was tested in the same study.The fold change in ALDOA mRNA, relative to saline, was calculated todetermine in vivo potency (“ND” indicates “not determined).

TABLE C Comparison of anti-miR-122 compound structure and potency Foldchange in ALDOA SEQ relative to saline Compound # Sequence and ChemistryID 1 mg/kg 3 mg/kg 10 mg/kg 38011C_(S)CAU_(S)TG_(S)U_(S)CAC_(S)AC_(S)TC_(S)C_(S)A 3 1.47 2.10 3.89 38012C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 1.79 4.69 4.57 38013C_(S)CAU_(S)TG_(S)TC_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 1.31 1.61 2.55 38014C_(S)CAU_(S)TG_(S)U_(S)CAC_(S)AC_(S)TC_(S)C_(S)A_(E) 3 1.16 1.82 2.9438015 ^(Me)C_(S)CAT_(S)TG_(S)T_(S)CA^(Me)C_(S)A^(Me)C_(S)T^(Me)C_(S)^(Me)C_(S)A_(E) 3 1.43 1.64 1.82 38016 ^(Me)C_(S)CAT_(S)TGT_(S)^(Me)C_(S)A^(Me)C_(S)A^(Me)C_(S)T^(Me)C_(S) ^(Me)C_(S)A_(E) 3 1.43 2.344.18 38021 ^(Me)C_(L)CAT_(L)TGT_(L)^(Me)C_(L)A^(Me)C_(L)A^(Me)C_(L)T^(Me)C_(L) ^(Me)C_(L)A_(E) 3 1.46 3.013.91 38872 C_(S)CAU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A 3 1.80 4.044.89 38910 ^(Me)CC_(S)AU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A_(E) 3ND 2.35 3.26

As can be seen in Table C, single changes in the placement of a sugarmoiety or nucleobase can have an impact on in vivo potency. For example,the only difference between 38872 and 38011 is the placement of a cEtsugar moiety, however the in vivo potency of 0011 is significantly lowerthan that of 38872, with a comparable level of ALDOA de-repressionreached only at the higher dose of 10 mg/kg of 38011 compared to the 3mg/kg dose for compound 38872. Compound 38021, relative to 38016, hasLNA in place of cEt sugar moieties, and has a similar potency to 38016,thus this difference did not impact potency. Of this group of compounds,compounds 38012, 38016, 38021 and 38872 were identified as activecompounds.

Additional studies were performed to evaluate certain additionalanti-miR-122 compounds. The results of these studies are shown in TableD. Compounds 38646, 38647, 38648, 38649, 38650, 38651, and 38652 weretested together in one in vivo study, and compounds 38659 and 38660 weretested together in another in vivo study.

TABLE D Comparison of anti-miR-122 compound structure and potency Foldchange in ALDOA Luciferase relative to SEQ mean saline Compound #Sequence and Structure ID EC₅₀ 3 mg/kg 10 mg/kg 38646 A_(E)^(Me)C_(E)A_(E) ^(Me)C_(E)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)-- 477.15 ND ND 38647 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)-- 4 57.44 2.61 4.8138648 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)-- 4 97.68 3.28 4.3638649 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)-- 4 46.76 2.844.46 38650 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)T_(E)GU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)-- 4 28.161.51 2.03 38651 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)T_(E)G_(E)U_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S--) 426.12 1.26 1.46 38652 ^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)-- 5 31.861.86 4.27 38659 C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E) 10130.01 4.44 4.82 38660 ^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E) 617.02 3.84 4.44

As above, these data illustrate that single changes to the placement ofa sugar moiety can have a substantial impact on in vivo potency.Further, it is shown that in vitro and in vivo potency are notnecessarily correlated. For example, compound 38659 has a low in vitropotency, but is a very potent inhibitor of miR-122 in vivo.

Comparisons of the anti-miR-122 compound structures and in vivo potencyrevealed an 11 nucleoside core sequence common to a group of activeanti-miR-122. This core sequence, where B-D-deoxy sugar moieties andbicyclic sugar moieties are in the same position on the anti-miR-122nucleotide sequence, is highlighted in Table D-2. The nucleobasesequence of the 11 nucleoside core is complementary to nucleobases 2 to12 of miR-122 (SEQ ID NO: 1).

TABLE D-2 Potent anti-miR-122 modified oligonucleotides with a commoncore sequence Cmpd # Position on miR- SEQUENCE (5′ to 3′) and STRUCTURESEQ ID 122 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 NO38012 C_(s) C A_(s) T T G U_(s) C_(s) C_(s) A C_(s) T C_(s) C_(s) A 338016 ^(Me)C_(s) C A T_(s) T G T_(s) T_(s) ^(Me)C_(s) ^(Me)C_(s) A_(E) 338021 C_(L) C A T_(L) T G T_(L) C_(L) C_(L) 4 38646 A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E) C A_(s) T T G U_(s) A C_(s) T C_(s) C_(s) 4 38647 A_(E)^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(s) T T G U_(s) C_(s) A C_(s) AC_(s) T C_(s) C_(s) 4 38648 A_(E) ^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E)A_(E) T T G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s) 4 38649 A_(E)^(Me)C_(E) A_(E) ^(Me)C_(E) ^(Me)C_(E) A_(E) T_(E) T G U_(s) C_(s) AC_(s) A C_(s) T C_(s) C_(s) 4 38652 ^(Me)C_(E) A_(E) A_(E) A_(E)^(Me)C_(E) A_(E) C_(s) C A_(s) T T G U_(s) C_(s) A C_(s) A C_(s) T C_(s)C_(s) 5 38659 C_(s) C A_(s) T T G U_(s) C_(s) A C_(s) A C_(s) T C_(s)C_(s) T_(E) 10 38660 ^(Me)C_(E) A_(E) A_(E) A_(E) ^(Me)C_(E) A_(E) C_(s)C A_(s) T T G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s) T_(E) 6 38872C_(s) C A U_(s) T G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s) A 3 38910^(Me)C_(E) C_(s) A U_(s) T G U_(s) C_(s) A C_(s) A C_(s) T C_(s) C_(s)A_(E) 3

These data illustrate the discovery of a certain core nucleoside patternthat yields a potent inhibitor of miR-122 in vivo.

HCV Replicon Studies

An HCV replicon assay was used to determine the ability of ananti-miR-122 compound to inhibit the replication of HCV, includingparent HCV genotypes and HCV genotypes with mutations that conferresistance to anti-viral agents. Compound 38649 was tested in thisassay, to determine its ability to inhibit the replication of HCVsub-genomic replicons of genotype 1a (H77 strain), genotype 1b, andseveral variants of genotype 1b (A156T, A156S, D168a, and V36M).

For this assay, the cell line used was the cell line ET, a Huh7 humanhepatoma cell line that contains a subgenomic replicon of HCV with astable luciferase reporter and three cell culture-adaptive mutations(luc-ubi-neo/ET). The luciferase reporter is used as an indirect measureof HCV replication. The HCV replicon antiviral evaluation assay examinedthe effects of the compound at six half-log concentrations of eachcompound. Human interferon alpha-2b was included as a positive controlcompound. Sub-confluent cultures of the ET line were plated into 96-wellplates and the next day anti-miR-122 compound was transfected into thecells with cationic lipid. Cells were processed 72 hours later when thecells were still sub-confluent. HCV replicon levels were assessed as HCVRNA replicon-derived luciferase activity. The EC₅₀ (concentration atwhich 50% inhibition was observed) was calculated for each HCV genotype,and is shown in Table E. The selectivity index (SI₅₀, a ratio of theEC₅₀ for viral replication to the EC₅₀ for innate cytotoxicity) was alsocalculated and is shown in Table E.

TABLE E Anti-Viral Activity of Compound 38649 Antiviral SelectivityActivity EC₅₀ Index HCV Genotype (nM) SI₅₀ HCV Genotype 1b 57.8 nM 4.0HCV Genotype 1b variant V36M 139.6 nM  >2.0 HCV Mutant A156S 45.9 nM 5.7HCV Mutant A156T 26.7 nM 10.0 HCV Mutant D168A 16.2 nM 12.0 HCV Genotype1a (H77 strain) 14.1 nM 15.0

The results from the replicon assay demonstrate anti-viral activity ofcompound 38649 against multiple HCV genotypes. The anti-viral activitywas sustained for the period of time for which the assay was performed(18 days). The activity of compound 38649 is similarly robust againstHCV replicons comprising mutations known to be resistant to certainprotease inhibitors prescribed to treat HCV infection.

Single Dose Studies of Anti-miR-122

Compound 38649 was tested in a single dose study in mice, to determinethe onset of action, maximal target derepression, and duration ofaction, at doses ranging from 0.3 mg/kg to 30 mk/kg. An ED₅₀ was alsocalculated from this study.

Anti-miR compound was administered intraperitoneally to groups of 5 miceeach, at doses of 0.3, 1.0, 3.0, 10, and 30 mg/kg. For the 0.3 and 1.0doses, groups of animals were sacrified at days 3, 7, and 28. For the3.0, 10 and 30 mg/kg doses, groups of animals were sacrified at days 3,5, 7, 14, 21, and 2838649. ALDOA mRNA levels in liver were measured byquantitative PCR, and compared to ALDOA mRNA levels in liver ofsaline-treated mice, to calculate the fold change in ALDOA expression.

As shown in FIG. 1A, ALDOA derepression was observed as early as day 3and maintained for more than 28 days after dosing of compound 38649.Maximal target derepression was achieved at 10 mg/kg. An ED₅₀ of 6.7mg/kg was calculated from the day 7 data (FIG. 1B).

Physicochemical Characteristics

Evaluation of physicochemical characteristics may include: measurementof viscosity, to determine whether a solution of the anti-miR issuitable for administration via certain types of parenteraladministration, for example subcutaneous administration; calculation ofanti-miR half life in liver, to estimate the frequency at which theanti-miR-122 compound could be administered in human subjects; andmetabolic stability assay, to identify compounds which may besusceptible to cleavage by nucleases.

Metabolic stability was evaluated by incubating anti-miR-122 compoundwith non-human primate liver lysate. Nuclease activity in the livertissue homogenate was confirmed by using reference oligonucleotides,which included a compound with known resistance to nuclease activity, acompound susceptible to 3′-exonuclease activity, and a compoundsusceptible to endonuclease activity. An internal standard compound wasused to control for extraction efficiency. At the 0 hour and 24 hourtime points, each sample was subjected to high-performance liquidchromatography time-of-flight mass spectrometry (HPLC-TOF MS) to measureoligonucleotide lengths and amounts. The percentage loss is determinedby comparing the amount of full-length compound at the 0 hour and 24hour time points. Compounds 38646, 38647, 38648, 38649, 38650, 38651,38652, 38659, and 38660 exhibited a percentage loss of 10% or less atthe 24 hour time point. Compound 38012 exhibited a percentage loss ofapproximately 50% at the 24 hour time point.

An additional single dose study was performed in mice, to estimate thehalf-life of compound 38649. The half-life in liver was estimated to beat least two weeks.

Safety

To assess various safety parameters, an in vivo study in rodents wasperformed for certain of the compounds described herein, to evaluate thepotential the compounds to trigger a pro-inflammatory response.Parameters assessed included changes in organ weights, such as spleenweight and liver weight, and the expression of interferon-induciblegenes, such as IFIT and OASL, in the liver. Serum chemistries were alsoevaluated. Additionally, for certain compounds, safety parameters wereevaluated in non-human primates and included hematological endpoints,serum chemistry, organ weights, coagulation, complement activation,cytokine/chemokine changes, and pro-inflammatory gene expression.

While the tested compounds exhibited some variability amongst thesaftety parameters evaluated, several of the compounds, includingcompound 38649, were found to have particularly suitable safetyprofiles.

Example 2 Conjugated Anti-miR-122 Modified Oligonucleotides

Anti-miR-122 modified oligonucleotides were conjugated to aGalNAc-containing moiety, to determine whether the conjugation wouldimprove the potency of the oligonucleotides.

GalNAc-containing compounds were formed by conjugating the structure inFIG. 2 to the 3′ end of the 38649 modified oligonucleotide. The linkagebetween the GalNAc-containing moiety and the 3′-end of 38649 varied, asshown in Table F-1. For example, in compound 38368, theGalNAc-containing moiety is linked directly to the 3′-terminalnucleoside of 38649 through a phosphodiester linkage, as shown in FIG.3C, where X is a phosphodiester linkage and MO is compound 38649. Incompound 38458, the GalNAc-containing moiety is linked to the3′-terminal nucleoside of 38649 through a β-D-deoxynucleoside, with aphosphorothioate linkage between the 3′-terminal nucleoside of 38649 anda phosphodiester linkage between the β-D-deoxynucleoside and theGalNAc-containing moiety, as shown in FIG. 3A, where X₂ is aphosphorothioate linkage, m is 1, N_(m) is a β-D-deoxynucleoside, X₁ isa phosphodiester linkage, and MO is compound 38649.

TABLE F-1 GalNAc-containing compounds Compound # Compound structure38368 Structure III of FIG. 3C, where X is a phosphodiester linkage andMO is compound 38649 38371 Structure III of FIG. 3C, where X is aphosphorothioate linkage and MO is compound 38649 38458 Structure I ofFIG. 3A, where X₂ is a phophorothioate linkage, m is 1, N_(m) is aβ-D-deoxynucleoside, X₁ is a phosphodiester linkage, and MO is compound38649 38459 Structure I of FIG. 3A, where X₂ is a phophodiester linkage,m is 1, N_(m) is a β-D-deoxynucleoside (dA), X₁ is a phosphodiesterlinkage, and MO is compound 38649 38597 Structure I of FIG. 3A, where X₂is a phosphothioate linkage, m is 1, N_(m) is a 2′-O-methoxyethylnucleoside, X₁ is a phosphodiester linkage, and MO is compound 3864938598 Structure I of FIG. 3A, where X₂ is a phophorothioate linkage, mis 1, N_(m) is a X₁ is a phosphodiester linkage, and MO is compound38649

The GalNAc-conjugated modified oligonucleotides were assessed for invivo potency, release of unconjugated modified oligonucleotide from theGalNAc-conjugated modified oligonucleotide, and liver and tissueconcentration.

Potency studies were conducted according to the protocol used toevaluate the unconjugated modified oligonucleotides, described above.Compound was injected into mice, and in vivo potency was assessed at day7 by measuring the de-repression of ALDOA. The dosages of conjugatedcompounds indicate the dosage of modified oligonucleotide administered.

As shown in FIG. 4, each of the three GalNAc-conjugated modifiedoligonucleotides tested was more potent than the unconjugated modifiedoligonucleotide. Compounds 38368 and 38371 exhibited an increase inpotency of approximately 3-fold, relative to unconjugated 38649 (FIG.4A). Compounds 38458 and 38459, each of which has aβ-D-deoxyribonucleoside linking group, exhibited at least a 10-foldincrease in potency (FIG. 4B). Compounds 38597 and 38598, each of whichhas a 2′-sugar modified linking group, also exhibited at least a 10-foldincrease in potency (FIG. 4C). In additional studies, potency increasesof up to 20-fold have been observed for compounds 38459, 38458, 38597,and 38598.

An additional experiment was conducted to include a wider range of dosesof compound 38459. Compound 38459 (n=6) or compound 38649 (n=3) wasadministered to mice, and ALDOA levels in liver and cholesterol levelsin blood were measured seven days later. Average ALDOA and cholesterollevels were calculated and are shown in Table F-2. As shown in TableF-2, a single, subcutaneous dose of compound 38459 exhibited increasedpotency relative to unconjugated compound 38649, with respect toincreasing ALDOA levels and lowering cholesterol levels. In thisexperiment, the calculated ED₅₀ for compound 38459 was 0.19 mg/kg, andthe calculated ED₅₀ for compound 38649 was 3.5 mg/kg (an 18-folddifference in potency).

TABLE F-2 Increased potency of conjugated anti-miR-122 compound ALDOACholesterol Compound Dose Fold change mg/dL 38649 (unconjugated) 1.0mg/kg 1.2 100.2 3.0 mg/kg 2.2 81.2  10 mg/kg 3.3 73.4 38459 (GalNAc-0.03 mg/kg  1.1 95.4 conjugated) 0.1 mg/kg 1.7 84.4 0.3 mg/kg 2.8 74   1mg/kg 3.5 59.2   3 mg/kg 3.8 61.8  10 mg/kg 3.8 61.4

Also measured was the amount of unconjugated modified oligonucleotide inthe liver and kidney tissue 7 days following a single subcutaneous doseof compounds 38368 and 38371 at doses of 1 mg/kg and 3 mg/kg, andcompounds 38458 and 38459 at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg.Each sample was subjected to high-performance liquid chromatographytime-of-flight mass spectrometry (HPLC-TOF MS) to measureoligonucleotide lengths and amounts. The lower limit of quantitation(LLOQ) by this method is 0.2-1.0 μg/g.

The GalNAc-conjugated modified oligonucleotides were found to havevarying rates of formation of unconjugated modified oligonucleotide. Forexample, following administration of compound 38368, less than 10% ofcompound 38649 (an unconjugated modified oligonucleotide) is detected inthe liver. Following administration of compound 38371, compound 38649was not detected in the liver at either dose of compound 38371.Conversely, seven days following subcutaneous administration of compound38459, the only unconjugated modified oligonucleotide species detectedwas unconjugated 38649; the parent compound 38459 was not detected.Following administration of compound 38458, unconjugated modifiedoligonucleotide was detected in two forms: 38649, as well as 38649-PO-A(a metabolite of compound 38458). This metabolite was detected at higherlevels than unconjugated 38649.

Also measured was the amount of unconjugated modified oligonucleotide inthe liver 24 hours following a single subcutaneous dose of compounds38458 and 38459 at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Anti-miRlevels were measured by LC-TOF. The lower limit of quantitation (LLOQ)by this method is 0.2-1.0 μg/g. It was observed that followingadministration of compound 38459, 90% of the total compound present inthe liver was unconjugated compound 38649. Following administration of38458, approximately 46% of total compound present in the liver wasunconjugated compound 38649. Thus, unconjugated compound 38649 isreleased more rapidly from compound 38459 than from compound 38458.These data suggest that the metabolism of the conjugated compound isinfluenced by the attachment between the linker and the modifiedoligonucleotide.

Oligonucleotides generally accumulate to the highest levels in kidneytissue, followed by liver tissue. To determine whether the GalNAcconjugate altered the accumulation of compound in liver tissue comparedto kidney tissue, relative to unconjugated compound, the amount ofunconjugated 38649 was also measured in the kidney tissue. As describedabove, following administration of compound 38459, 100% of the totalcompound found in the liver is unconjuated 38649, indicating completerelease of 38649 from the GalNAc-conjugated compound 38459. Followingadministration of compound 38459, compound 38649 accumulated less in thekidney compared to the liver, (i.e. exhibited a lower kidney:liverratio), relative to accumulation of compound 38649 followingadministration of compound 38649. Thus, compound 38459 canpreferentially deliver compound 38649 to the liver, while minimizingdelivery to the kidney, as compared to unconjugated 38649.

The onset and duration of action for compound 38459 was evaluated in anin vivo study. Groups of mice were given a single, subcutaneous (SC)dose of compound 38459 at 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Anadditional group of mice was administered compound 38649 at a dose of 10mg/kg. A group of animals from each treatment was sacrificed on each ofdays 1, 2, 3, 4, 5, 6, 14, 21, 28, and 56. RNA was isolated from liverand ALDOA mRNA levels were measured by real-time PCR. The mean ALDOAlevel for each group was calculated. The fold change relative to thecontrol group (PBS-treated) is shown in Table G.

TABLE G Onset and duration of action of compound 38459 Days followingFold change in ALDOA single 38459 38459 38459 38459 38649 SC dose 3mg/kg 1 mg/kg 0.3 mg/kg 0.1 mg/kg 10 mg/kg 1 4.9 3.6 1.7 1.4 2.2 2 4.23.2 2.4 1.4 4.7 3 4.4 4.6 3.5 1.6 3.4 4 5.1 4.9 3.3 2.2 4.6 5 5.9 4.93.9 2.1 4.5 6 5.1 4.5 3.2 2.2 3.6 14 4.8 4.3 3.4 1.7 3.1 21 5.9 4.9 4.02.2 3.6 28 4.8 4.7 2.9 2.0 4.2 56 5.6 4.6 2.6 1.7 3.2

The data in Table G demonstrate that compound 38459, as well as compound38649, has a rapid onset of action, as evidenced by ALDOA derepressionas early as 1 day following a single dose of compound. Further, ALDOAderepression is maintained for at least 8 weeks following a single doseof compound.

These data demonstrate that the GalNAc-conjugated compound 38459, whichis at least 10-fold more potent than the unconjugated 38649 compound,achieves this potency at significantly lower liver tissueconcentrations, with preferential delivery to the liver tissue.Additionally, compound 38459 exhibits a rapid onset of action, and aduration of action of at least 8 weeks.

Also tested were LNA-containing unconjugated and conjugated modifiedoligonucleotides, shown in Table H.

TABLE H LNA-containing compounds SEQ ID Compound # Sequence (5′ to 3′)and Modifications Structure NO 36848C_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L), Unconjugated 7 36852C_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) Conjugated as in 7Structure III of FIG. 3C, where X is PO and MO is 36848 36632C_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L) Conjugated as in 7Structure I of FIG. 3A, where X₂ is a phophodiester linkage, m is 1,N_(m) is a β-D- deoxynucleoside (dA), X₁ is a phosphodiester linkage,and MO is compound 36848

Sugar and linkage moietes are indicated as follows: where nucleosidesnot followed by a subscript indicate β-D-deoxyribonucleosides;nucleosides followed by a subscript “L” indicate LNA nucleosides; andeach internucleoside linkage is a phosphorothioate internucleosidelinkage.

Compounds 36848 and 36852 were tested for in vivo potency according tothe same protocol as described above, to evaluate the ability of thecompounds to inhibit miR-122 activity and increase ALDOA expression.While each compound was a potent inhibitor of miR-122, theGalNAc-conjugated compound 36852 exhibited greater potency thanunconjugated compound 36848 (approximately 3-fold greater).

Compound 36632 was also tested for in vivo potency in a single doseadministration study, following a similar protocol as described above,at doses of 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, and10.0 mg/kg. Compound 36632 demonstrated fold increases in ALDOAexpression of 1.6, 2.7, 3.7, 4.3, 4.7, 6.0, respectively, relative toPBS-treated control. Compound 36848, at doses of 1.0 mg/kg, 3.0 mg/kg,and 10 mg/kg resulted in fold increases in ALDOA expression of 1.6, 2.5,and 5.3, respectively. A comparison of compound 36632 to compound 36848revealed an increase in potency of approximately 30-fold for theconjugated compound, relative to the unconjugated compound.

Example 3 Mouse Model of HCV Infection

Due to host-pathogen specificity, HCV can only infect humans andchimpanzees. As such, smaller species, such as mice, that are typicallyused for experimental in vivo studies cannot be infected with HCV fortesting of candidate agents for the treatment of HCV infection. Toaddress this problem, human liver chimeric mouse models may be utilized(see, e.g.,, Bissig et al., Proc Natl Acad Sci USA, 2007,104:20507-20511; Bissig et al., J Clin Invest., 2010, 120: 924-930). Inthis model, the livers of immunodeficient mice are repopulated withhuman hepatocytes, resulting in a chimeric liver in which most of thehepatocytes are human hepatocytes. The mice are then infected with HCVand treated with anti-HCV agents. This mouse model is commerciallyavailable from, for example, PhoenixBio.

Anti-miR-122 compounds are tested in mice with human chimeric liversthat have been infected with HCV. Groups of animals (n=5-10) receive oneor more doses of anti-miR-122 compound, e.g., at a dose identified fromthe treatment regimen study. For pharmacokinetic analyses andmeasurement of HCV RNA levels, plasma is collected at varioustimepoints. Liver tissue is collected when the study is terminated.

In some embodiments, inhibition of miR-122 is confirmed by measuringhuman ALDOA mRNA levels. It is expected that administration of ananti-miR-122 compound reduces HCV RNA levels in the serum of the mouse.

Example 4 HCV RNA Level Reduction in Response to miR-122 Inhibition

A human chimeric mouse liver model was used to evaluate the effects ofmiR-122 inhibition on miR-122 target gene expression and HCV viraltiter.

Human Chimeric Liver Mice

The effects of miR-122 inhibition on target gene expression wereevaluated in human chimeric liver mice without HCV infection. Groups ofmice (n=6) were treated with a single dose of PBS, 0.3 mg/kg, 1.0 mg/kg,3.0 mg/kg, or 10 mg/kg of compound 38459. Seven days followingtreatment, the study was terminated and liver tissue was collected formeasurement of ALDOA expression and compound tissue concentration. ALDOAmRNA levels were increased relative to ALDOA mRNA levels in PBS-treatedmice, however the derepression of ALDOA expression was 3-fold to 5-foldless than that observed in wild-type mice. Compound 38459 levels wereapproximately 3-fold lower in chimeric liver mice, relative toconcentrations in wild-type mice. These observations are consistent withthe reduced expression of the asialoglycoprotein receptor (ASGPR) in thehuman chimeric liver mice, relative to wild-type mice. As theaccumulation of compound in the liver cell is dependent upon uptake bythe ASGPR, a reduced expression of ASGPR would be expected to result inreduced accumulation of GalNAc-conjugated modified oligonucleotide, andconsequently reduced sensitivity to the ability of compound 38459 tode-repress endogenous targets of miR-122, such as ALDOA. Accordingly,the human chimeric liver mouse model may underpredict the activity ofcompound 38459 in a subject where ASGPR expression is maintained.Preliminary data suggest that ASGPR expression is maintained at similarlevels in livers of HCV-infected patients relative to livers of non-HCVinfected subjects.

Treatment of HCV-Infected Human Chimeric Liver Mice

Anti-miR-122 compounds were tested in a human chimeric liver mouse modelof HCV infection. The livers of immunodeficient mice were repopulatedwith human hepatocytes, resulting in a chimeric liver in which most ofthe hepatocytes are human hepatocytes. Approximately 3.5 weeks followinginoculation with HCV genotype 1a, mice with an HCV RNA level of >1×10⁶copies/ml were selected for inclusion in this study (Day −7).

For a single week study, a group of 3 animals was treated with a single10 mg/kg dose of 38459 on Day 0. Blood was collected on Day −7, 0, 3,and 7. The study was terminated on day 7, when in addition to blood,liver tissue and kidney tissue were collected. In this study, HCV RNAlevels were reduced at Days 3 and 7.

For a multiple week study, groups of 5 animals each were treated asfollows: PBS (n=5); 3 mg/kg 38459 (n=5); 10 mg/kg 38459 (n=4-5); or 30mg/kg 38459 (n=4-5). An additional group of animals was treated with 10mg/kg unconjugated compound 36848 (n=5). Treatment was administered as asingle, subcutaneous injection on Day 0. Blood was collected on Days −7,0, 3, 7, 10, 14, 17, 21, 24, 28, and 35. HCV RNA levels in blood weremeasured by real-time PCR according to routine methods, and are shown inTable I. Unless otherwise indicated, each treatment group contained 5animals. As shown in Table I, HCV RNA levels were signficantly reducedas early as Day 3 in the groups treated with 10 mg/kg or 30 mg/kg ofcompound 38459, which reduction was sustained through at least Day 35.Statistical significance was calculated by 2way ANOVA analysis of meanHCV RNA levels in compound-treated animals, normalized to mean HCV RNAlevels in PBS-treated animals. In this study, unconjugated compound36848 did not reduce HCV RNA levels. These results are also illustratedin graphic form in FIG. 5A.

TABLE I GalNAc-conjugated anti-miR-122 reduces HCV titer 36848 3845938459 38459 Day PBS Average 10 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg −72.66E+08 2.90E+08 2.54E+08 2.76E+08 2.60E+08 0 2.08E+08 2.92E+083.26E+08 2.38E+08 2.70E+08 3 1.97E+08 3.20E+08 2.90E+08 8.10E+07*4.76E+07**** 7 1.65E+08 3.26E+08 1.76E+08 3.16E+07**** 1.22E+07**** 101.59E+08 2.74E+08 1.21E+08 2.70E+07**** 7.52E+06**** 14 1.19E+082.02E+08 9.34E+07 2.37E+07**** 4.82E+06**** 17 1.67E+08 2.10E+089.68E+07 2.94E+07**** 4.89E+06**** 21 1.49E+08 2.36E+08 9.72E+073.06E+07**** 7.65E+06**** (n = 4) 24 1.43E+08 2.14E+08 8.46E+073.35E+07**** 7.95E+06**** (n = 4) 28 1.43E+08 1.63E+08 8.48E+074.16E+07*** 1.13E+07**** (n = 4) 31 1.37E+08 1.99E+08 9.22E+07 5.18E+07*1.98E+07**** (n = 4) (n = 4) 35 1.44E+08 1.88E+08 1.03E+08 5.80E+07*2.35E+07**** (n = 4) (n = 4) ****p < .0001; ***p < 0.0005; *p < 0.05

These results demonstrate that, following a single administration ofGalNAc-conjugated modified oligonucleotide 38459, HCV viral titer wassignificantly reduced in HCV-infected animals, with an early onset andsustained duration of action.

An additional study was performed to evaluate the effects of compound38459 in the human chimeric liver mouse model of HCV infection, wherethe mice are infected with HCV genotype 3a. Groups of 5 animals eachwere treated as follows: PBS (n=4); 10 mg/kg 38459 (n=5); or 30 mg/kg38459 (n=5). Mice were inoculated with HCV genotype 3a. Seven days priorto treatment, blood was collected from mice for measurement of viraltiter. Treatment was administered as a single, subcutaneous injection onDay 0. Blood was collected on Days 0, 3, 7, 10, 14, 17, 21, 24, and 28following treatment. HCV RNA levels in blood were measured by real-timePCR according to routine methods. As shown in FIG. 5B, HCV RNA levelswere signficantly reduced early as Day 3 in the groups treated with 10mg/kg or 30 mg/kg of compound 38459, and this reduction was sustainedthrough at least Day 28.

Also observed was a substantial reduction in steatosis in the livers ofthe mice treated with compound 38459. The reduced steatosis was observedin mice infected with HCV, and in uninfected mice, suggesting thatinhibition of miR-122 can reduce steatosis both in the presence andabsence of HCV infection.

Example 5 Conjugated Shorter Modified Oligonucleotides

GalNAc-containing compounds were formed by conjugating a structure inFIG. 3 to the 3′ end of the modified oligonucleotides shown in Table J.Sugar moieties, internucleoside linkages, and nucleobases are indicatedas follows: nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “S” areS-cEt nucleosides; and each internucleoside linkage is aphosphorothioate internucleoside linkage.

TABLE J Unconjugated and Conjugated Modified Oligonucleotides SEQSequence and Modifications Structure ID NO 38591U_(s)TGU_(s)C_(s)AC_(s)AC_(s)TC_(s)C_(s)A_(s) Unconjugated 8 38633U_(s)TGU_(s)C_(s)AC_(s)AC_(s)TC_(s)C_(s)A_(s) Structure I of FIG. 3A,where X₂ is a 8 phophodiester linkage, m is 1, N_(m) is a β-D-deoxynucleoside (dA), X₁ is a phosphodiester linkage 38998C_(s)A_(s)C_(s)A_(s)C_(s)U_(s)C_(s)C_(s) Unconjugated 9 38634C_(s)A_(s)C_(s)A_(s)C_(s)U_(s)C_(s)C_(s) Structure I of FIG. 3A, whereX₂ is a 9 phophodiester linkage, m is 1, N_(m) is a β-D- deoxynucleoside(dA), X₁ is a phosphodiester linkage

To determine in vivo potency, the compounds were evaluated for theirability to de-repress the expression of liver aldolase A (ALDOA).Compounds were administered to mice, and ALDOA mRNA levels weremeasured, by quantitative PCR, in RNA isolated from liver. The foldchange in ALDOA mRNA, relative to saline, was calculated to determine invivo potency (FIGS. 6A and 6B and 7A and 7B). The ED50 (concentration ofcompound at which ALDOA derepression is 50% of maximum) and ED90(concentration of compound at which ALDOA deprepression is 90% ofmaximum) calculated from the results of those experiments are shown inTable K and L.

TABLE K In vivo potency of conjugated and unconjugated anti-miR-122compounds Fold Compound ED50 (mg/kg) Fold change ED90 (mg/kg) changeExperiment 1 (FIG. 6A) 38634 0.03 456 0.3 212 38998 13.7 63.8 Experiment2 (FIG. 6B) 38634 0.04 290 0.43 99.3 38998 11.6 42.7

TABLE L In vivo potency of conjugated and unconjugated anti-miR-122compounds Fold Compound ED50 (mg/kg) Fold change ED90 (mg/kg) changeExperiment 1 (FIG. 7A) 38633 0.08 27 0.25 26 38591 2.2 6.62 Experiment 2(FIG. 7B) 38633 0.15 20 0.94 10 38591 3.0 8.9

As shown in Table K, GalNAc conjugation according to the presentinvention improved the ED₅₀ and ED₉₀ of an 8-mer anti-miR-122 compoundby at least 100-fold. As shown in Table L, GalNAc conjugation accordingto the present invention improved the ED₅₀ and ED₉₀ of a 13-meranti-miR-122 compound by at least 10-fold.

Derepression of another miR-122 target gene, CD320, was also determinedfor compounds 38634 and 38998. The results were similar to the resultsobtained for ALDOA shown in Table K: GalNAc conjugation according to thepresent invention improved the ED₅₀ by 343-fold and 272-fold inexperiments 1 and 2, respectively, and improved the ED₉₀ by 492-fold and545-fold in experiments 1 and 2, respectively.

GalNAc conjugation described herein also improved cholesterol-loweringpotency was also observed for the compounds comprising GalNAc. Exemplaryresults from experiment 1 are shown in FIGS. 8A and 8B. Compounds 38633and 38634, which are GalNAc conjugates, were more potent than compounds38591 and 38998, which lack GalNAc. Similar results were obtained forexperiment 2 (data not shown).

Example 6 Pharmacodynamic Activity of Anti-miR-122 Compounds inNon-Human Primates

Anti-miR-122 compounds were tested in normal non-human primates(cynomolgus monkeys). A single dose of GalNAc-conjugated compound 38459or unconjugated compound 38649 was administered subcutaneously (n=3 foreach compound). PBS was administered as a control treatment (n=5). Onday 4 and day 8 following administration of compound, liver tissue wascollected, and RNA was isolated for measurement of ALDOA levels. Totalcholesterol in blood was measured on day 8. As shown in Table L, ALDOAderepression is observed at day 4 and day 8, at each dose of compound38459, including the lowest dose of 1 mg/kg. Cholesterol lowering wasalso observed with the lowest dose of compound 38459. Thus,GalNAc-conjugated compound 38459 is significantly more potent innon-human primates, relative to unconjugated compound 38649.Additionally, both compounds have a duration of action of at least oneweek following a single dose in non-human primates.

TABLE L Inhibition of miR-122 in non-human primates ALDOA (Day 4) ALDOA(Day 8) Cholesterol Treatment fold change fold change (Day 8) mg/dL PBS1.0 95.3 38649, 100 mg/kg 3.4 4.0 67.0 38459, 1 mg/kg 5.0 3.9 64.338459, 10 mg/kg 3.0 3.6 66.7 38459, 100 mg/kg 4.0 4.1 65.3

Example 7 Pharmacokinetic Activity of Conjugated Anti-miR-122 Compounds

The plasma and tissue pharmacokinetics of anti-miR-122 compounds wereevaluated in mice and non-human primates.

A single, subcutaneous dose of compound 38649 or GalNAc-conjugatedcompound 38459 was administered to CD-1 mice. Blood was collected amultiple time points over a 24 hour period following administration, andthe total amount of compound in the blood was measured byhybridization-based ELISA.

A single, subcutaneous dose of compound 38649 or GalNAc-conjugatedcompound 38459 was administered to non-human primates. Blood wascollected at multiple time points over a 24 hour period followingadministration, and the total amount of compound in the blood wasmeasured by LC-MS.

As shown in FIG. 9, in mouse (FIG. 9A) and non-human primates (FIG. 9B),GalNAc-conjugated compound 38459 is cleared more rapidly from plasma,compared to unconjugated compound 38649. Following administration ofGalNAc-conjugated compound 38459, unconjugated compound 38649 is notdetected, indicating that conjugated compound 38459 is not metabolizedin the blood (data not shown)

In this study, tissue levels of compounds were also measured in theliver and kidney of mice (Table M) and non-human primates (Table N).

TABLE M Compound tissue levels in mice 24 hours after single doseCompound Administered: 38459 (+GalNAc) 38649 Tissue: Kidney Liver KidneyLiver Compound Mean Mean K/L Mean Mean K/L Dose detected (μg/g) (μg/g)Ratio (μg/g) (μg/g) Ratio 1 mg/ 38649 1.1 5.7 0.19 18.4 4 4.6 kg Total1.1 7.4 0.15 compound 3 mg/ 38649 8.2 15.8 0.52 83.9 10.8 7.6 kg Total16.8 27.7 0.61 compound

TABLE N Compound tissue levels in non-human primates 72 hours aftersingle dose Compound Administered: 38459 (+GalNAc) 38649 Tissue: KidneyLiver Kidney Liver Compound Mean Mean K/L Mean Mean K/L Dose detected(μg/g) (μg/g) Ratio (μg/g) (μg/g) Ratio  1 mg/kg 38649 5.6 27.2 0.21Total 31.3 34 0.92 compound  10 mg/kg 38649 124 148 0.84 283.3 61.2 4.6Total 513.5 186.3 2.7 compound 100 mg/kg 38649 374.1 418.8 0.89 1430242.3 5.9 Total 2129.1 547.2 3.9 compound

Following administration, compound 38459 is rapidly metabolized tounconjugated compound 38649 in liver and kidney. Additionally,consistent with the data from the mouse study described above, thekidney to liver ratio of compound 38459 is significantly lower than thatof compound 38649.

Based on the concentration of compound in the liver 24 hours followingadministration, it was estimated that approximately 6 μg/g ofGalNAc-conjugated compound 38459 and approximately 30 μg/g ofunconjugated compound 38459 results in 90% maximal potency at day 7 (asmeasured by ALDOA derepression). Thus, compound 38459 results in greaterpotency at a lower liver tissue concentration, relative to unconjugatedcompound 38649.

These data demonstrate that in non-human primates and mice, conjugationto a GalNAc-containing moiety results in significantly enhanced deliveryof modified oligonucleotide to the liver. Further, a low ED₅₀ coupledwith a lower kidney to liver ratio suggests that GalNAc-conjugatedcompound 38459 may have a high therapeutic index.

Example 8 Toxicology and safety studies of anti-miR-122 compounds

Multiple studies were conducted in mice, rodents and non-human primates,to evaluate the safety and tolerability of GalNAc-conjugated compound38459.

For example, compound 38459 was evaluated in a pro-inflammatory study inrats. Male Sprague Dawley rats were administered a single, subcutaneousdose of compound 38459. At day 14 following administration, expressionof ALDOA and CXCL13 (an interferon-inducible gene) was measured inliver.

As shown in Table 0, no increase in CXCL13 expression was detected at adose as high as 100 mg/kg, while ALDOA levels were elevated starting atthe 1 mg/kg dose. A known inflammatory anti-miR-122 compound was alsotested, and resulted in increases of CXCL13 levels of 2- to 2.5-fold atthe 10, 30 and 100 mg/kg doses.

TABLE O Compound 38459 does not increase pro-inflammatory geneexpression Dose of compound ALDOA CXCL13 38459 Fold-change Fold-change0.1 mg/kg  1.2 1.4 0.3 mg/kg  1.6 1.6  1 mg/kg 2.5 1.5  3 mg/kg 2.9 0.810 mg/kg 2.8 0.6 30 mg/kg 3.2 0.8 100 mg/kg  3.4 0.7

Additional toxicology studies were conducted in mice and non-humanprimates (cynomolgus monkeys), and no significant adverse effects wereobserved at therapeutically relevant doses.

Example 9 Conjugated Shorter Modified Oligonucleotides

Cholesterol-containing compounds were formed by conjugating cholesterolto the 3′ end of the modified oligonucleotides shown in Table P. Sugarmoieties, internucleoside linkages, and nucleobases are indicated asfollows: nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “S” areS-cEt nucleosides; and each internucleoside linkage is aphosphorothioate internucleoside linkage, except the internucleosidelinkages indicated by subscript (0), which are phosphodiester linkages.

TABLE P Unconjugated and Conjugated Modified Oligonucleotides Sequenceand SEQ ID Modifications Structure NO 38998 CsAsCsAsCsUsCsCsUnconjugated 9 38070 CsAsCsAsCsUsCsCs

9 MO is CsAsCsAsCsUsCsCs

To determine in vivo potency, the compounds were evaluated for theirability to de-repress the expression of liver aldolase A (ALDOA).Compounds were administered to mice, and ALDOA mRNA levels weremeasured, by quantitative PCR, in RNA isolated from liver. The foldchange in ALDOA mRNA, relative to saline, was calculated to determine invivo potency. The ED50 (concentration of compound at which ALDOAderepression is 50% of maximum) and ED90 (concentration of compound atwhich ALDOA deprepression is 90% of maximum) calculated from the resultsof those experiments are shown in Table Q.

TABLE Q In vivo potency of conjugated and unconjugated anti-miR-122compounds ED50 Fold ED90 Fold Compound (mg/kg) change (mg/kg) change38070 0.08 78.8 1.27 31.6 38998 6.3 40.1

As shown in Table Q, cholesterol conjugation according to the presentinvention improved the ED₅₀ and ED₉₀ of an 8-mer anti-miR-122 compoundby at least 30-fold.

Derepression of another miR-122 target gene, CD320, was also determinedfor compounds 38070 and 38998. The results were similar to the resultsobtained for ALDOA (data not shown).

Cholesterol conjugation described herein also improvedcholesterol-lowering potency. At most concentrations tested, compound38070 reduced cholesterol to a greater extent than the sameconcentration of compound 38998 (data not shown).

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, GENBANK®accession numbers, and the like) cited in the present application isspecifically incorporated herein by reference in its entirety.

1. A compound comprising a modified oligonucleotide consisting of 16 to22 linked nucleosides, wherein the nucleobase sequence of the modifiedoligonucleotide is complementary to miR-122 (SEQ ID NO: 1) and whereinthe modified oligonucleotide comprises at least 16 contiguousnucleosides of the following nucleoside pattern I in the 5′ to 3′orientation:(R)_(X)-N^(Q)-N^(Q)-N^(B)-N^(B)-N^(Q)-N^(B)-N^(Q)-N^(B)-N^(Q)-N^(B)-N^(B)-(N^(Z))_(y)wherein each R is, independently, a non-bicyclic nucleoside or abicyclic nucleoside; X is from 4 to 10; each N^(B) is, independently, abicyclic nucleoside; each N^(Q) is, independently, a non-bicyclicnucleoside; Y is 0 or 1; and N^(Z) is a modified nucleoside or anunmodified nucleoside.
 2. The compound of claim 1, wherein the modifiedoligonucleotide comprises at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, or 22 contiguous nucleosides ofnucleoside pattern I.
 3. The compound of claim 1 wherein each bicyclicnucleoside is independently selected from an LNA nucleoside, a cEtnucleoside, and an ENA nucleoside.
 4. (canceled)
 5. (canceled)
 6. Thecompound of claim 1, wherein each bicyclic nucleoside is a cEtnucleoside. 7.-11. (canceled)
 12. The compound of claim 1 wherein eachnon-bicyclic nucleoside is independently selected from aβ-D-deoxyribonucleoside, a β-D-ribonucleoside, 2′-O-methyl nucleoside, a2′-O-methoxyethyl nucleoside, and a 2′-fluoronucleoside.
 13. Thecompound of claim 1 wherein each non-bicyclic nucleoside isindependently selected from a β-D-deoxyribonucleoside, and a2′-O-methoxyethyl nucleoside. 14.-19. (canceled)
 20. The compound ofclaim 1, wherein X is 4, 5, 6, 7, 8, 9, or
 10. 21. The compound of claim1, wherein Y is 0 or
 1. 22. (canceled)
 23. The compound of claim 1wherein: a. X is 7; each R is a 2′-O-methoxyethyl nucleoside; each N^(B)is an S-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; and Yis 0; b. X is 4; (R)_(X) is N^(R1)-N^(R2)-N^(R3)-N^(R4), wherein each ofN^(R1) and N^(R3) is a S-cEt nucleoside and each of N^(R2) and N^(R4) isa β-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; Y is 1; and N^(Z) is aβ-D-deoxyribonucleoside c. X is 4; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4), wherein each of N^(R1) and N^(R4) is aS-cEt nucleoside and each of N^(R2) and N^(R3) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; Y is 1; and N^(Z) is a 2′-O-methoxyethylnucleoside d. X is 7; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), and N^(R4) and is a 2′-O-methoxyethylnucleoside, each of N^(R5) and N^(R7) is a β-D-deoxyribonucleoside, andN^(R6) is S-cEt nucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; and Y is 0; e. X is 7; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), N^(R4), and N^(R5) is a 2′-O-methoxyethylnucleoside, N^(R6) is S-cEt nucleoside, and N^(R7) is aβ-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; each N^(Q)is a β-D-deoxyribonucleoside; and Y is 0; f. X is 7; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7), wherein each ofN^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is 2′-O-methoxyethylnucleoside, and N^(R7) is a β-D-deoxyribonucleoside; each N^(B) is anS-cEt nucleoside; each N^(Q) is a β-D-deoxyribonucleoside; and Y is 0;g. X is 10; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7)-N^(R8)-N^(R9)-N^(R10),wherein each of N^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is2′-O-methoxyethyl nucleoside, each of N^(R7) and N^(R9) is a an S-cEtnucleoside; each of N^(R8) and N^(R10) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; and Y is 0; h. X is 10; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4)-N^(R5)-N^(R6)-N^(R7)-N^(R8)-N^(R9)-N^(R10),wherein each of N^(R1), N^(R2), N^(R3), N^(R4), N^(R5), and N^(R6) is2′-O-methoxyethyl nucleoside, each of N^(R7) and N^(R9) is a an S-cEtnucleoside; and each of N^(R8) and N^(R10) is a β-D-deoxyribonucleoside;each N^(B) is an S-cEt nucleoside; each N^(Q) is aβ-D-deoxyribonucleoside; Y is 1 and N_(Z) is a 2′-O-methoxyethylnucleoside; i. X is 4; (R)_(X) is N^(R1)-N^(R2)-N^(R3)-N^(R4), whereineach of N^(R1) and N^(R4) is an S-cEt nucleoside, and each of N^(R1) andN^(R3) is a β-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside;each N^(Q) is a β-D-deoxyribonucleoside; Y is 1 and N^(Z) is aβ-D-deoxyribonucleoside; j. X is 4; (R)_(X) isN^(R1)-N^(R2)-N^(R3)-N^(R4), wherein N^(R1) is a 2′-O-methoxyethylnucleoside, each of N^(R2) and N^(R4) is an S-cEt nucleoside, and N^(R3)is a β-D-deoxyribonucleoside; each N^(B) is an S-cEt nucleoside; eachN^(Q) is a β-D-deoxyribonucleoside; Y is 1 and N^(Z) is a2′-O-methoxyethyl nucleoside.
 24. The compound of claim 1, wherein thenucleobase sequence of the modified oligonucleotide is at least 90%complementary to the nucleobase sequence of miR-122 (SEQ ID NO: 1). 25.The compound of claim 1, wherein at least one internucleoside linkage isa modified internucleoside linkage.
 26. The compound of claim 1, whereinthe nucleobase sequence of the modified oligonucleotide is selected fromSEQ ID NOs: 3 to 6, wherein each T is independently selected from T andU.
 27. (canceled)
 28. The compound of claim 1 having the structure: a.A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S); b.C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A; c.^(Me)C_(S)CAT_(S)TGT_(S) ^(Me)C_(S)A^(Me)C_(S)A^(Me)C_(S)T^(Me)C_(S)^(Me)C_(S)A_(E); d. A_(E) ^(Me)C_(E)A_(E)^(Me)C_(E)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S); e. A_(E)^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S); f. A_(E)^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S); g.^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S) h.^(Me)C_(E)A_(E)A_(E)A_(E)^(Me)C_(E)A_(E)C_(S)CA_(S)TTGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)T_(E); i.C_(S)CAU_(S)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A; or j.^(Me)C_(E)C_(S)AU_(S)TGU_(s)C_(S)AC_(S)AC_(S)TC_(S)C_(S)A_(E) whereinthe superscript “Me” indicates 5-methylcytosine; nucleosides notfollowed by a subscript are β-D-deoxyribonucleosides; nucleosidesfollowed by a subscript “E” are 2′-MOE nucleosides; nucleosides followedby a subscript “S” are S-cEt nucleosides; and each internucleosidelinkage is a phosphorothioate internucleoside linkage.
 29. The compoundof claim 1, wherein the compound comprises a conjugate moiety linked tothe 5′ terminus or the 3′ terminus of the modified oligonucleotide.30.-32. (canceled)
 33. The compound of claim 29, wherein the conjugatemoiety comprises at least one ligand selected from a carbohydrate,cholesterol, a lipid, a phospholipid, an antibody, a lipoprotein, ahormone, a peptide, a vitamin, a steroid, and a cationic lipid.
 34. Thecompound of claim 29, wherein the compound has the structure:L_(n)-linker-MO wherein each L is, independently, a ligand and n is from1 to 10; and MO is a modified oligonucleotide.
 35. The compound of claim29, wherein the compound has the structure:L_(n)-linker-X-MO wherein each L is, independently, a ligand and n isfrom 1 to 10; X is a phosphodiester linkage or a phosphorothioatelinkage; and MO is a modified oligonucleotide.
 36. The compound of claim29, wherein the compound has the structure:L_(n)-linker-X₁-N_(m)-X₂-MO wherein each L is, independently, a ligandand n is from 1 to 10; each N is, independently, a modified orunmodified nucleoside and m is from 1 to 5; X₁ and X₂ are each,independently, a phosphodiester linkage or a phosphorothioate linkage;and MO is a modified oligonucleotide.
 37. (canceled)
 38. (canceled) 39.The compound of claim 36, wherein if n is greater than 1, L_(n)-linkerhas the structure:L-Q′

-S-Q″

_(n)- wherein each L is, independently, a ligand; n is from 1 to 10; Sis a scaffold; and Q′ and Q″ are, independently, linking groups.
 40. Thecompound of claim 39, wherein Q′ and Q″ are each independently selectedfrom a peptide, an ether, polyethylene glycol, an alkyl, a C₁-C₂₀ alkyl,a substituted C₁-C₂₀ alkyl, a C₂-C₂₀ alkenyl, a substituted C₂-C₂₀alkenyl, a C₂-C₂₀ alkynyl, a substituted C₂-C₂₀ alkynyl, a C₁-C₂₀alkoxy, a substituted C₁-C₂₀ alkoxy, amino, amido, a pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and 6-aminohexanoic acid.
 41. The compound ofclaim 39, wherein the scaffold links 2, 3, 4, or 5 ligands to a modifiedoligonucleotide.
 42. (canceled)
 43. The compound of claim 36, whereinthe compound has the structure:

wherein: B is selected from —O—, —S—, —N(R^(N))—, —Z—P(Z′)(Z″)O—,—Z—P(Z′)(Z″)O—N_(m)—X—, and —Z—P(Z′)(Z″)O—N_(m)—Y—; MO is a modifiedoligonucleotide; R^(N) is selected from H, methyl, ethyl, propyl,isopropyl, butyl, and benzyl; Z, Z′, and Z″ are each independentlyselected from O and S; each N is, independently, a modified orunmodified nucleoside; m is from 1 to 5; X is selected from aphosphodiester linkage and a phosphorothioate linkage; Y is aphosphodiester linkage; and the wavy line indicates the connection tothe rest of the linker and ligand(s).
 44. (canceled)
 45. The compound ofclaim 36, wherein n is from 1 to
 5. 46. (canceled)
 47. The compound ofclaim 36, wherein at least one ligand is a carbohydrate.
 48. Thecompound of claim 36, wherein at least one ligand is selected frommannose, glucose, galactose, ribose, arabinose, fructose, fucose,xylose, D-mannose, L-mannose, D-galactose, L-galactose, D-glucose,L-glucose, D-ribose, L-ribose, D-arabinose, L-arabinose, D-fructose,L-fructose, D-fucose, L-fucose, D-xylose, L-xylose,alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose,beta-D-mannopyranose, alpha-D-glucofuranose, Beta-D-glucofuranose,alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-galactofuranose,beta-D-galactofuranose, alpha-D-galactopyranose, beta-D-galactopyranose,alpha-D-ribofuranose, beta-D-ribofuranose, alpha-D-ribopyranose,beta-D-ribopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose,glucosamine, galactosamine, sialic acid, N-acetylgalactosamine. 49.(canceled)
 50. The compound of claim 36, wherein each ligand isN-acetylgalactosamine.
 51. The compound of claim 36, wherein thecompound has the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide.
 52. A compound comprising a modifiednucleotide and a conjugate moiety, wherein the modified oligonucleotidehas the structure C_(L)CA_(L)TTG_(L)T_(L)CAC_(L)AC_(L)TC_(L)C_(L),wherein the subscript “L” indicates an LNA and nucleosides not followedby a subscript are β-D-deoxyribonucleosides, and each internucleosidelinkage is a phosphorothioate internucleoside linkage, and wherein theconjugate moiety is linked to the 3′ terminus of the modifiedoligonucleotide and has the structure:

wherein each N is, independently, a modified or unmodified nucleosideand m is from 1 to 5; X₁ and X₂ are each, independently, aphosphodiester linkage or a phosphorothioate linkage; and MO is amodified oligonucleotide.
 53. The compound of claim 51, wherein at leastone of X₁ and X₂ is a phosphodiester linkage.
 54. The compound of claim51, wherein each of X₁ and X₂ is a phosphodiester linkage.
 55. Thecompound of claim 36, wherein m is 1, 2, 3, 4, or
 5. 56. (canceled) 57.The compound of claim 36, wherein N_(m) is N′_(p)N″, wherein each N′ is,independently, a modified or unmodified nucleoside and p is from 0 to 4;and N″ is a nucleoside comprising an unmodified sugar moiety.
 58. Thecompound of claim 57, wherein p is 0, 1, 2, 3, or
 4. 59. (canceled) 60.The compound of claim 57, wherein each N′ comprises an unmodified sugarmoiety. 61.-65. (canceled)
 66. The compound of claim 57, wherein N″ is aβ-D-deoxyriboadenosine or a β-D-deoxyriboguanosine.
 67. (canceled) 68.(canceled)
 69. The compound of claim 36 wherein the sugar moiety of eachN is independently selected from a β-D-ribose, a β-D-deoxyribose, a2′-O-methoxy sugar, a 2′-O-methyl sugar, a 2′-fluoro sugar, and abicyclic sugar moiety. 70.-72. (canceled)
 73. A compound comprising amodified nucleotide and a conjugate moiety, wherein the modifiedoligonucleotide has the structure A_(E) ^(Me)C_(E)A_(E) ^(Me)C_(E)^(Me)C_(E)A_(E)T_(E)TGU_(S)C_(S)AC_(S)AC_(S)TC_(S)C_(S), whereinnucleosides not followed by a subscript are β-D-deoxyribonucleosides,nucleosides followed by a subscript “E” are 2′-MOE nucleosides,nucleosides followed by a subscript “S” are S-cEt nucleosides, and eachinternucleoside linkage is a phosphorothioate internucleoside linkage;and wherein the conjugate moiety is linked to the 3′ terminus of themodified oligonucleotide and has the structure:

wherein X is a phosphodiester linkage; m is 1; N is aβ-D-deoxyriboadenosine; Y is a phosphodiester linkage; and MO is themodified oligonucleotide.
 74. (canceled)
 75. A method of inhibiting theactivity of miR-122 in a cell comprising contacting a cell with acompound of claim
 1. 76. (canceled)
 77. (canceled)
 78. A methodcomprising administering to an HCV-infected subject a compound ofclaim
 1. 79.-114. (canceled)